Tobacco nicotine demethylase genomic clone and uses thereof

ABSTRACT

The present invention features tobacco nicotine demethylase nucleic acid and amino acid sequences, tobacco plants and plant components containing such sequences, including tobacco plants and plant components having reduced expression or altered enzymatic activity of nicotine demethylase, methods of use of nicotine demethylase sequences to create plants having altered levels of nornicotine or N′-nitrosonornicotine (“NNN”) or both relative to a control plant, as well as tobacco articles having reduced levels of nornicotine or NNN.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. ProvisionalApplication No. 60/665,451, filed Mar. 24, 2005, U.S. ProvisionalApplication No. 60/665,097, filed Mar. 24, 2005, U.S. ProvisionalApplication No. 60/646,764, filed Jan. 25, 2005, U.S. ProvisionalApplication No. 60/607,357, filed Sep. 3, 2004, and U.S. ProvisionalApplication No. 60/566,235, filed Apr. 29, 2004.

The present application is also a divisional of U.S. application Ser.No. 11/110,062, filed Apr. 19, 2005, now U.S. Pat. No. 7,700,851, whichis a continuation-in-part of PCT/US2004/034065, filed Oct. 15, 2004,which claims the benefit of U.S. Application No. 60/556,235, filed Apr.29, 2004, and which is a continuation-in-part of U.S. application Ser.No. 10/934,944, filed Sep. 3, 2004, which, in turn, is acontinuation-in-part of U.S. application Ser. No. 10/686,947, filed Oct.16, 2003. U.S. application Ser. No. 11/110,062, filed Apr. 19, 2005, isalso a continuation-in-part of PCT/US2004/034218, filed Oct. 15, 2004,which claims the benefit of U.S. Application No. 60/566,235, filed Apr.29, 2004, and which is a continuation-in-part of U.S. application Ser.No. 10/943,507, filed Sep. 17, 2004, and which is also acontinuation-in-part application of U.S. application Ser. No. 10/934,944filed, Sep. 3, 2004 and U.S. application Ser. No. 10/943,507, filed Sep.17, 2004, each of which is a continuation-in-part of U.S. applicationSer. No. 10/686,947, filed Oct. 16, 2003, which, in turn, claims thebenefit of U.S. Provisional Application No. 60/503,989, filed Sep. 18,2003, U.S. Provisional Application No. 60/485,368, filed Jul. 8, 2003,and U.S. Provisional Application No. 60/418,933, filed Oct. 16, 2002.U.S. application Ser. No. 10/686,947, filed Oct. 16, 2003 is acontinuation-in-part of U.S. application Ser. No. 10/387,346, filed Mar.12, 2003, which, in turn, is a continuation-in-part of U.S. applicationSer. No. 10/340,861, filed Jan. 10, 2003, which, in turn, is acontinuation-in-part of U.S. application Ser. No. 10/293,252, filed Nov.13, 2002, which claims the benefit of U.S. Provisional Application No.60/363,684, filed Mar. 12, 2002, U.S. Provisional Application No.60/347,444, filed Jan. 11, 2002, and U.S. Provisional Application No.60/337,684, filed on Nov. 13, 2001. All of the applications listed areincorporated herein by reference.

BACKGROUND

Cytochrome p450s catalyze enzymatic reactions for a diverse range ofchemically dissimilar substrates that include the oxidative,peroxidative, and reductive metabolism of endogenous and xenobioticsubstrates. In plants, p450s participate in biochemical pathways thatinclude the synthesis of plant products such as phenylpropanoids,alkaloids, terpenoids, lipids, cyanogenic glycosides, and glucosinolates(Chappell, Annu Rev. Plant Physiol. Plant Mol. Biol. 46:521-547, 1995).Cytochrome p450s, also known as p450 heme-thiolate proteins, usually actas terminal oxidases in multi-component electron transfer chains, calledp450-containing monooxygenase systems. Specific reactions catalyzed bythese enzyme systems include demethylation, hydroxylation, epoxidation,N-oxidation, sulfooxidation, N-, S-, and 0-dealkylations, desulfation,deamination, and reduction of azo, nitro, and N-oxide groups.

The diverse role of Nicotiana plant p450 enzymes has been implicated ineffecting a variety of plant metabolites such as phenylpropanoids,alkaloids, terpenoids, lipids, cyanogenic glycosides, glucosinolates,and a host of other chemical entities. Some p450 enzymes can impact thecomposition of plant metabolites. For example, it has been long desiredto improve the flavor and aroma of certain plants by altering a plant'sprofile of selected fatty acids through breeding; however very little isknown about mechanisms involved in controlling the levels of these leafconstituents. The down regulation or up regulation of p450 enzymesassociated with the modification of fatty acids may facilitateaccumulation of desired fatty acids that provide more preferred leafphenotypic qualities.

The function of p450 enzymes and their broadening roles in plantconstituents is still being discovered. For instance, a special class ofp450 enzymes was found to catalyze the breakdown of fatty acid intovolatile C6- and C9-aldehydes and 13-alcohols that are majorcontributors of “fresh green” odor of fruits and vegetables. The levelof other novel targeted p450s may be altered to enhance the qualities ofleaf constituents by modifying lipid composition and related breakdownmetabolites in Nicotiana leaf. Several of these constituents in leaf areaffected by senescence that stimulates the maturation of leaf qualityproperties. Still other reports have shown that p450s enzymes are play afunctional role in altering fatty acids that are

involved in plant-pathogen interactions and disease resistance.

In other instances, p450 enzymes have been suggested to be involved inalkaloid biosynthesis. As provided in patent applications by Applicant,from which the present application claims priority, and which areincorporated by reference herein, nornicotine, a minor alkaloid found inNicotiana tabacum, is produced by the p450-mediated demethylation ofnicotine followed by acylation and nitrosation at the N position therebyproducing a series of N-acylnonicotines and N-nitrosonornicotines.N-demethylation, catalyzed by a p450 demethylase, is thought to be aprimary source of nornicotine biosynthesis in Nicotiana.

There exists a need in the art for reagents and methods for modifyingplant phenotypes. In particular, there exists a need for reagents andmethods for modifying nicotine demethylase. The present inventionprovides a number of strategies for modifying expression of a nicotinedemethylase.

SUMMARY OF THE INVENTION

The present inventors have identified and characterized a genomic cloneof nicotine demethylase from tobacco. Included herein are sequences forthe nicotine demethylase protein coding region, the 3′ untranslatedregion (“3′ UTR”), a single intron, and the nicotine demethylase genepromoter along with its transcriptional regulatory sequences (FIG. 1).Further described is the use of these sequences to create transgenicplants having altered levels of nornicotine or N′-nitrosonornicotine(“NNN”) or both relative to a control plant.

Accordingly, in the first aspect, the invention features an isolatednucleic acid molecule, for example, a DNA sequence, containing anucleotide sequence encoding a nicotine demethylase. In desirableembodiments, the nucleotide sequence of the first aspect issubstantially identical to a nucleotide sequence encoding a tobacconicotine demethylase, such as a tobacco nicotine demethylase containinga nucleotide sequence that is at least 70% identical to the nucleotidesequence of SEQ ID NO:1 or SEQ ID NO:3, or that contains nucleotides2010-2949 and/or 3947-4562 of SEQ ID NO:1, or that contains the sequenceof SEQ ID NO:1 or SEQ ID NO:3. The isolated nucleic acid molecule of thefirst aspect of the invention, for example, is operably linked to apromoter functional in a plant cell and desirably is contained in anexpression vector. In other desirable embodiments, the expression vectoris contained in a cell, e.g., a plant cell. Desirably, the plant cell,such as a tobacco plant cell, is included in a plant. In anotherdesirable embodiment, the invention features a seed, e.g., a tobaccoseed, from a plant containing the expression vector, where the seedincludes an isolated nucleic acid molecule that hybridizes understringent conditions to the sequence of SEQ ID NO:3 operably linked to aheterologous promoter sequence. Furthermore, the invention features aplant derived from a germinated seed containing the expression vector, aleaf, either green or cured, from the plant, and an article ofmanufacture made from the leaf.

In another desirable embodiment, the nucleotide sequence contains asequence that hybridizes under stringent conditions to the complement ofthe nucleotide sequence of SEQ ID NO:1 and/or SEQ ID NO:3, or to afragment of SEQ ID NO:1 or SEQ ID NO:3. Desirably, the nucleotidesequence encodes a nicotine demethylase that is substantially identicalto the amino acid sequence of SEQ ID NO:2. In a further desirableembodiment of the first aspect of the invention, the nicotinedemethylase has at least 70% amino acid sequence identity to thenicotine demethylase amino acid sequence of SEQ ID NO:2 or to a fragmentof a nicotine demethylase having altered (e.g., reduced) enzymaticactivity as compared to the full-length polypeptide. Desirably, thenicotine demethylase includes the amino acid sequence of SEQ ID NO:2.

In another aspect, the invention features an isolated nucleic acidmolecule containing a promoter that hybridizes under stringentconditions to the sequence of SEQ ID NO:6, or a fragment thereof thatdrives transcription. Desirably, the promoter (i) is induced followingtreatment with ethylene or during senescence; and (ii) includes (a) basepairs 1-2009 of SEQ ID NO:1, or (b) at least 200 consecutive base pairsidentical to 200 consecutive base pairs of the sequence defined by basepairs 1-2009 of SEQ ID NO:1, or (c) a 20 base pair nucleotide portionidentical in sequence to a 20 consecutive base pair portion of thesequence set forth in base pairs 1-2009 of SEQ ID NO:1.

A further aspect of the invention features an isolated nucleic acidpromoter containing a nucleotide sequence having 50% or more sequenceidentity with the sequence of SEQ ID NO:6.

Desirably, this isolated nucleic acid promoter is induced followingtreatment with ethylene or during senescence and, for example, includesthe sequence of SEQ ID NO:6. Alternatively, the promoter may include afragment obtainable from SEQ ID NO:6, where the fragment drivestranscription of a heterologous gene or reduces or alters nicotinedemethylase enzymatic activity (for example, silences gene expression).In a desirable embodiment the promoter sequence is operably linked to aheterologous nucleic acid sequence, and may, for example be contained inan expression vector. In other desirable embodiments the expressionvector is contained in a cell, e.g., a plant cell. Desirably, the plantcell, such as a tobacco plant cell, is included in a plant. In anotherdesirable embodiment, the invention features a seed, e.g., a tobaccoseed, from a plant containing the expression vector, where the seedincludes an isolated nucleic acid molecule that hybridizes understringent conditions to the sequence of SEQ ID NO:6 operably linked to aheterologous nucleic acid sequence. Furthermore, the invention featuresa plant derived from a germinated seed containing the promoter of thisaspect of the invention, a leaf, either green or cured, from the plant,and an article of manufacture made from the leaf.

Another aspect of the invention features a method of expressing aheterologous gene in a plant. This method involves (i) introducing intoa plant cell a vector containing a promoter sequence having 50% or moresequence identity with the sequence of SEQ ID NO:6 operably linked to aheterologous nucleic acid sequence; and (ii) regenerating a plant fromthe cell. In addition, this method may involve sexually transmitting thevector to progeny and, further, may include the step of collecting theseed produced by the progeny.

In yet another aspect, the invention features a method of reducingexpression of nicotine demethylase in a tobacco plant. This methodincludes the steps of (i) introducing into the tobacco plant a vectorcontaining the sequence of SEQ ID NO:6 or a fragment obtainable from SEQID NO:6 operably linked to a heterologous nucleic acid sequence and (ii)expressing the vector in the tobacco plant. In a desirable embodiment ofthis method, expression of the nicotine demethylase is silenced. Inother desirable embodiment, the vector expresses RNA, such as antisenseRNA or an RNA molecule capable of inducing RNA interference (RNAi).

In a further desirable aspect, the invention features an isolatednucleic acid molecule containing an intron that hybridizes understringent conditions to the sequence of SEQ ID NO:5, or a fragmentthereof that reduces or alters nicotine demethylase enzymatic activity(for example, silences gene expression) or can serve as a molecularmarker to identify nicotine demethylase nucleic acid sequences. In adesirable embodiment, the intron includes (a) base pairs 2950-3946 ofSEQ ID NO:1, or (b) at least 200 consecutive base pairs identical to 200consecutive base pairs of the sequence defined by base pairs 2950-3946of SEQ ID NO:1, or (c) a 20 base pair nucleotide portion identical insequence to a 20 consecutive base pair portion of the sequence set forthin base pairs 2950-3946 of SEQ ID NO:1.

Another desirable aspect of the invention features an isolated nucleicacid intron including a nucleotide sequence having 50% or more sequenceidentity with the sequence of SEQ ID NO:5, or a fragment thereof thatreduces or alters nicotine demethylase enzymatic activity (for example,silences gene expression) or can serve as a molecular marker to identifynicotine demethylase nucleic acid sequences. Silencing gene expressionmay, for example, involve homologous recombination or a mutation thatresults in a gene product that does not have nicotine demethylaseactivity. In particular, the intron may include the sequence of SEQ IDNO:5 or a fragment obtainable from SEQ ID NO:5. Desirably, an isolatednucleic acid molecule including an intron is operably linked to aheterologous nucleic acid sequence and this sequence desirably isincluded in an expression vector. In another embodiment, the expressionvector is contained in a cell, such as a plant cell. In particular, thecell may be a tobacco cell. A plant, e.g., a tobacco plant, including aplant cell plant containing the sequence of SEQ ID NO:5 or a fragmentobtainable from SEQ ID NO:5 operably linked to a heterologous nucleicacid sequence in an expression vector is another desirable embodiment ofthe present invention. Further, a seed, for example, a tobacco seed,from a plant, where the seed contains an intron that hybridizes understringent conditions to SEQ ID NO:5 operably linked to a heterologousnucleic acid sequence is also desirable. Furthermore, the inventionfeatures a plant derived from the germinated seed containing the intronof this aspect of the invention, a leaf, either green or cured, from theplant, and an article of manufacture made from the green or cured leaf.

A further aspect of the invention features a method of expressing anintron in a plant. This method involves (i) introducing into a plantcell an expression vector containing the sequence of SEQ ID NO:5 or afragment obtainable from SEQ ID NO:5 operably linked to a heterologousnucleic acid sequence; and (ii) regenerating a plant from the cell. In adesirable embodiment, this method also involves (iii) sexuallytransmitting the vector to progeny, and may include the additional stepof collecting the seed produced by the progeny. The method desirablyincludes, for example, regenerating a plant from the germinated seed, aleaf, either green or cured, from the plant, and a method of making anarticle of manufacture from the leaf.

In yet another aspect, the invention features a method of reducingexpression of nicotine demethylase in a tobacco plant. This methodincludes the steps of (i) introducing into the tobacco plant a vectorcontaining the sequence of SEQ ID NO:5 or a fragment obtainable from SEQID NO:5 operably linked to a heterologous nucleic acid sequence and (ii)expressing the vector in the tobacco plant. In a desirable embodiment ofthis method, expression of the nicotine demethylase is silenced. Inother desirable embodiment, the vector expresses RNA, such as antisenseRNA or an RNA molecule capable of inducing RNA interference (RNAi).

In an additional aspect, the invention features an isolated nucleic acidmolecule containing an untranslated region that hybridizes understringent conditions to the sequence of SEQ ID NO:7 or a fragmentthereof that can alter the expression pattern of a gene, reduces oralters nicotine demethylase enzymatic activity (for example, silencesgene expression), or can be used as a marker to identify nicotinedemethylase nucleic acid sequences. In a desirable embodiment of thisaspect of the invention, the untranslated region includes (a) base pairs4563-6347 of SEQ ID NO:1, or (b) at least 200 consecutive base pairsidentical to 200 consecutive base pairs of the sequence defined by basepairs 4563-6347 of SEQ ID NO:1, or (c) a 20 base pair nucleotide portionidentical in sequence to a 20 consecutive base pair portion of thesequence set forth in base pairs 4563-6347 of SEQ ID NO:1.

An additional desirable aspect of the invention features an isolatednucleic acid untranslated region containing a nucleotide sequence having50% or more sequence identity with the sequence of SEQ ID NO:7.Desirably, the untranslated region includes the sequence of SEQ ID NO:7or the untranslated region includes a fragment obtainable from SEQ IDNO:7 that can alter the expression pattern of a gene, reduces or altersnicotine demethylase enzymatic activity (for example, silences geneexpression), or can be used as a marker to identify nicotine demethylasenucleic acid sequences. The untranslated region desirably is operablylinked to a heterologous nucleic acid sequence and may be contained inan expression vector. Further, this expression vector is desirablycontained in a cell, such as a plant cell, e.g., a tobacco cell. Anotherdesirable embodiment of the invention features a plant, such as atobacco plant, including a plant cell containing a vector that includesan isolated nucleic acid sequence that has 50% or more sequence identitywith the sequence of SEQ ID NO:7 and is operably linked to aheterologous nucleic acid sequence.

The invention also features a seed, for example, a tobacco seed, from aplant, where the seed includes an untranslated region that hybridizesunder stringent conditions to SEQ ID NO:7 operably linked to aheterologous nucleic acid sequence. Furthermore, the invention featuresa plant derived from a germinated seed containing the untranslatedregion of this aspect of the invention, a leaf, either green or cured,from the plant, and an article of manufacture made from the green orcured leaf.

In a further aspect, the invention features a method of expressing anuntranslated region in a plant. This method involves (i) introducinginto a plant cell a vector containing an isolated nucleic acid sequencethat has 50% or more sequence identity with the sequence of SEQ ID NO: 7and is operably linked to a heterologous nucleic acid sequence; and (ii)regenerating a plant from the cell. In addition, this method may alsoinvolve (iii) sexually transmitting the vector to progeny, anddesirably, includes the additional step of collecting the seed producedby the progeny. The method desirably includes regenerating a plant fromthe germinated seed, a leaf, either green or cured, from the plant, anda method of making an article of manufacture made from the green orcured leaf.

Furthermore, the invention features a method of reducing expression oraltering the enzymatic activity of nicotine demethylase in a tobaccoplant. This method includes the steps of (i) introducing into thetobacco plant a vector containing an isolated nucleic acid sequence thathas 50% or more sequence identity with the sequence of SEQ ID NO:7 andis operably linked to a heterologous nucleic acid sequence and (ii)expressing the vector in the tobacco plant. Desirably, expression of thenicotine demethylase is silenced. In other desirable embodiments thevector expresses RNA, e.g., antisense RNA or an RNA molecule capable ofinducing RNA interference (RNAi).

Another aspect of the invention features an expression vector includinga nucleic acid molecule containing a nucleotide sequence encoding anicotine demethylase, where the vector is capable of directingexpression of the nicotine demethylase encoded by the isolated nucleicacid molecule. Desirably, the vector includes the sequence of SEQ IDNO:1 or SEQ ID NO:3. In other desirable embodiments, the inventionfeatures a plant or plant component, e.g., a tobacco plant or plantcomponent (e.g., a tobacco leaf or stem), that includes a nucleic acidmolecule containing a nucleotide sequence encoding a polypeptide thatdemethylates nicotine.

A further aspect of the invention features a cell containing an isolatednucleic acid molecule that includes a nucleotide sequence encoding anicotine demethylase. Desirably this cell is a plant cell or a bacterialcell, such as an Agrobacterium.

Another aspect of the invention features a plant or plant component(e.g., a tobacco leaf or stem) containing an isolated nucleic acidmolecule that encodes a nicotine demethylase, where the nucleic acidmolecule is expressed in the plant or the plant component. Desirably,the plant or plant component is an angiosperm, a dicot, a solanaceousplant, or a species of Nicotiana. Other desirable embodiments of thisaspect are a seed or a cell from the plant or plant component, as wellas a leaf, either green or cured, derived from the plant and an articleof manufacture made therefrom.

In an additional aspect, the invention features a tobacco plant havingreduced expression of a nucleic acid sequence encoding a polypeptide,for example, one that includes the sequence of SEQ ID NO:2, and thatdemethylates nicotine, where the reduced expression (or a reduction inenzymatic activity) reduces the level of nornicotine in the plant. In adesirable embodiment, the tobacco plant is a transgenic plant, such asone that includes a transgene that, when expressed in the transgenicplant, silences gene expression of an endogenous tobacco nicotinedemethylase.

In particular, the transgenic plant desirably includes one or more ofthe following: a transgene that expresses an antisense molecule of atobacco nicotine demethylase or an RNA molecule capable of inducing RNAinterference (RNAi); a transgene that, when expressed in the transgenicplant, co-suppresses expression of a tobacco nicotine demethylase; atransgene that encodes a dominant negative gene product, e.g., a mutatedform the amino acid sequence of SEQ ID NO:2; a point mutation in a genethat encodes the amino acid sequence of SEQ ID NO:2; a deletion in agene that encodes a tobacco nicotine demethylase; and an insertion in agene that encodes a tobacco nicotine demethylase.

In other desirable embodiments, reduced expression of a nucleic acidsequence encoding a polypeptide occurs at the transcriptional level, atthe translational level, or at the post-translational level.

Another aspect of the invention features a tobacco plant containing arecombinant expression cassette stably integrated into the genomethereof, where the cassette is capable of effecting a reduction innicotine demethylase activity. Seeds of this tobacco plant are featuredin a desirable embodiment. Other desirable embodiments include leaf,either green or cured, derived from this plant and an article ofmanufacture made therefrom.

A further aspect of the invention features a method of expressing atobacco nicotine demethylase in a plant. This method involves (i)introducing into a plant cell an expression vector including a nucleicacid molecule containing a nucleotide sequence encoding a nicotinedemethylase; and (ii) regenerating a plant from the cell. In a desirableembodiment, this method features sexually transmitting the vector toprogeny, and desirably also includes the additional step of collectingthe seed produced by the progeny. Additional desirable embodimentsinclude a plant derived from the germinated seed, a leaf, either greenor cured, from the plant, and an article of manufacture made from thegreen or cured leaf.

An additional aspect of the invention features a substantially puretobacco nicotine demethylase. Desirably, this tobacco nicotinedemethylase includes an amino acid sequence having at least 70% identityto the amino acid sequence of SEQ ID NO:2 or includes the amino acidsequence of SEQ ID NO:2. In a desirable embodiment, the tobacco nicotinedemethylase, upon expression in a plant cell, converts nicotine tonornicotine. In other desirable embodiments, the tobacco nicotinedemethylase, upon expression in a plant cell, is predominantly localizedin leaves, or the tobacco nicotine demethylase is induced by ethylene oris expressed during plant senescence.

In a further aspect, the invention features a substantially pureantibody that specifically recognizes and binds to a tobacco nicotinedemethylase. Desirably, the antibody recognizes and binds to arecombinant tobacco nicotine demethylase, e.g., one containing thesequence of SEQ ID NO:2 or a fragment thereof.

Another aspect of the invention features a method of producing a tobacconicotine demethylase. This method involves the steps of: (a) providing acell transformed with an isolated nucleic acid molecule containing anucleotide sequence encoding a polypeptide that demethylates nicotine;(b) culturing the transformed cell under conditions for expressing theisolated nucleic acid molecule; and (c) recovering the tobacco nicotinedemethylase. The invention also features a recombinant tobacco nicotinedemethylase produced according to this method.

In an additional aspect, the invention features a method of isolating atobacco nicotine demethylase or fragment thereof. This method involvesthe steps of: (a) contacting the nucleic acid molecule of SEQ. ID NOS:1,3, 5, 6, or 7 or a portion thereof with a nucleic acid preparation froma plant cell under hybridization conditions providing detection ofnucleic acid sequences having at least 70% or greater sequence identityto the nucleic acid sequence of SEQ ID NOS:1, 3, 5, 6, or 7; and (b)isolating the hybridizing nucleic acid sequences.

In a further aspect, the invention features another method of isolatinga tobacco nicotine demethylase or fragment thereof. This method includesthe steps of: (a) providing a sample of plant cell DNA; (b) providing apair of oligonucleotides having sequence identity to a region of anucleic acid molecule having the sequence of SEQ ID NOS:1, 3, 5, 6, or7; (c) contacting the pair of oligonucleotides with the plant cell DNAunder conditions suitable for polymerase chain reaction-mediated DNAamplification; and (d) isolating the amplified tobacco nicotinedemethylase or fragment thereof. In a desirable embodiment of thisaspect, the amplification step is carried out using a sample of cDNAprepared from a plant cell. In another desirable embodiment, the tobacconicotine demethylase encodes a polypeptide which is at least 70%identical to the amino acid sequence of SEQ ID NO:2.

A further aspect of the invention features a method for reducing theexpression of tobacco nicotine demethylase in a plant or plantcomponent. This method involves the steps of: (a) introducing into plantcells a transgene encoding a tobacco nicotine demethylase operablylinked to a promoter functional in the plant cells to yield transformedplant cells; and (b) regenerating a plant or plant component from thetransformed plant cells, where the tobacco nicotine demethylase isexpressed in the cells of the plant or plant component, thereby reducingthe expression of tobacco nicotine demethylase in a plant or plantcomponent. In particular embodiments of this aspect of the invention,the transgene encoding the tobacco nicotine demethylase isconstitutively expressed or inducibly expressed, for example, in atissue-specific, cell-specific, or organ-specific manner. In anotherembodiment of this aspect of the invention, expression of the transgeneco-suppresses the expression of an endogenous tobacco nicotinedemethylase.

A further aspect of the invention features another method for reducingthe expression of tobacco nicotine demethylase in a plant or plantcomponent. This method includes the steps of: (a) introducing into plantcells a transgene encoding an antisense coding sequence of a tobacconicotine demethylase or an RNA molecule capable of inducing RNAinterference (RNAi) operably linked to a promoter functional in theplant cells to yield transformed plant cells; and (b) regenerating aplant or plant component from the transformed plant cells, where theantisense or an RNA molecule capable of inducing RNA interference (RNAi)of the coding sequence of the tobacco nicotine demethylase is expressedin the cells of the plant or plant component, thereby reducing theexpression of tobacco nicotine demethylase in a plant or plantcomponent. Desirably, the transgene encoding an antisense sequence or anRNA molecule capable of inducing RNA interference (RNAi) of a tobacconicotine demethylase is constitutively expressed or is induciblyexpressed, for instance in a tissue-specific, cell-specific, ororgan-specific manner.

An additional aspect of the invention features yet another method forreducing the expression of tobacco nicotine demethylase in a plant orplant component. This method involving the steps of: (a) introducinginto plant cells a transgene encoding a dominant negative gene productof a tobacco nicotine demethylase operably linked to a promoterfunctional in the plant cells to yield transformed plant cells; and (b)regenerating a plant or plant component from the transformed plantcells, where the dominant negative gene product of the tobacco nicotinedemethylase is expressed in the cells of the plant or plant component,thereby reducing the expression of tobacco nicotine demethylase in aplant or plant component. In particular embodiments of this aspect ofthe invention, the transgene encoding the dominant negative gene productis constitutively expressed or is inducibly expressed, for example, in atissue-specific, cell-specific, or organ-specific manner.

A further aspect of the invention features an additional method forreducing the expression or the enzymatic activity of tobacco nicotinedemethylase in plant cell. This method involves reducing the level of anendogenous tobacco nicotine demethylase, or its enzymatic activity, inthe plant cell. Desirably, the plant cell is from a dicot, a solanaceousplant, or a species of Nicotiana. In desirable embodiments of thisaspect, reducing the level of endogenous tobacco nicotine demethylaseinvolves expressing a transgene encoding an antisense nucleic acidmolecule or an RNA molecule capable of inducing RNA interference (RNAi)of a tobacco nicotine demethylase in the plant cell, or involvesexpressing a transgene encoding a double-stranded RNA molecule of atobacco nicotine demethylase in the plant cell. Desirably, thedouble-stranded RNA is an RNA sequence corresponding to the sequence ofSEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, or afragment thereof. In an additional embodiment, reducing the level ofendogenous tobacco nicotine demethylase involves co-suppression of theendogenous tobacco nicotine demethylase in the plant cell or involvesexpressing a dominant negative gene product in the plant cell. Inparticular, the dominant negative gene product may include a gene thatencodes a mutated form the amino acid sequence of SEQ ID NO:2.

In other desirable embodiments of this aspect of the invention, theendogenous tobacco nicotine demethylase includes a point mutation in agene that encodes the amino acid sequence of SEQ ID NO:2. In otherdesirable embodiments reducing the level of expression of an endogenoustobacco nicotine demethylase involves a deletion in a gene that encodesa tobacco nicotine demethylase or involves an insertion in a gene thatencodes a tobacco nicotine demethylase. The reduced expression may occurat the transcriptional level, at the translational level, or at thepost-translational level.

A further aspect of the invention features a method for identifying acompound which alters the expression of a tobacco nicotine demethylasein a cell. This method involves the steps of: (a) providing a cellcontaining a gene encoding a tobacco nicotine demethylase; (b) applyinga candidate compound to the cell; and (c) measuring expression of thegene encoding the tobacco nicotine demethylase, where an increase ordecrease in expression relative to an untreated control sample is anindication that the compound alters expression of the tobacco nicotinedemethylase.

In a desirable embodiment of this method, the gene of part (a) encodes atobacco nicotine demethylase having at least 70% identity to the aminoacid sequence of SEQ ID NO:2. Desirably, the compound decreases orincreases expression of the gene that encodes the tobacco nicotinedemethylase.

In another aspect, the invention features another method for identifyinga compound which alters the activity of a tobacco nicotine demethylasein a cell. This method involves the steps of: (a) providing a cellexpressing a gene encoding a tobacco nicotine demethylase; (b) applyinga candidate compound to the cell; and (c) measuring the activity of thetobacco nicotine demethylase, where an increase or decrease in activityrelative to an untreated control sample is an indication that thecompound alters activity of the tobacco nicotine demethylase. In adesirable embodiment of this aspect of the invention, the gene of step(a) encodes a tobacco nicotine demethylase having at least 70% identityto the amino acid sequence of SEQ ID NO:2. Desirably, the compounddecreases or increases the activity of the tobacco nicotine demethylase.

A further aspect of the invention features a cured tobacco plant orplant component containing (i) a reduced levels of nicotine demethylaseor (ii) a nicotine demethylase having an altered enzymatic activity anda reduced amount of a nitrosamine. Desirably, the plant component is atobacco leaf or tobacco stem. In a desirable embodiment, the nitrosamineis nornicotine, and the content of nornicotine desirably is less than 5mg/g, 4.5 mg/g, 4.0 mg/g, 3.5 mg/g, 3.0 mg/g, more desirably less than2.5 mg/g, 2.0 mg/g, 1.5 mg/g, 1.0 mg/g, more desirably less than 750μg/g, 500 μg/g, 250 μg/g, 100 μg/g, even more desirably less than 75μg/g, 50 μg/g, 25 μg/g, 10 μg/g, 7.0 μg/g, 5.0 μg/g, 4.0 μg/g, and evenmore desirably less than 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, 0.4 μg/g, 0.2μg/g, 0.1 μg/g, 0.05 μg/g, or 0.01 μg/g or wherein the percentage ofsecondary alkaloids relative to total alkaloid content therein is lessthan 90%, 70%, 50%, 30%, 10%, desirably less than 5%, 4%, 3%, 2%, 1.5%,1%, and more desirably less than 0.75%, 0.5%, 0.25%, or 0.1%. In anotherdesirable embodiment, the nitrosamine is N′-nitrosonornicotine (NNN),and the content of N′-NNN desirably is less than 5 mg/g, 4.5 mg/g, 4.0mg/g, 3.5 mg/g, 3.0 mg/g, more desirably less than 2.5 mg/g, 2.0 mg/g,1.5 mg/g, 1.0 mg/g, more desirably less than 750 μg/g, 500 μg/g, 250μg/g, 100 μg/g, even more desirably less than 75 μg/g, 50 μg/g, 25 μg/g,10 μg/g, 7.0 μg/g, 5.0 μg/g, 4.0 μg/g, and even more desirably less than2.0 μg/g, 1.0 μg/g, 0.5 μg/g, 0.4 μg/g, 0.2 μg/g, 0.1 μg/g, 0.05 μg/g,or 0.01 μg/g or wherein the percentage of secondary alkaloids relativeto total alkaloid content contained therein is less than 90%, 70%, 50%,30%, 10%, desirably less than 5%, 4%, 3%, 2%, 1.5%, 1%, and moredesirably less than 0.75%, 0.5%, 0.25%, or 0.1%. In additional desirableembodiments of this aspect of the invention, the cured tobacco plant orplant component is a dark tobacco, Burley tobacco, flue-cured tobacco,air-cured tobacco, or oriental tobacco.

Further, the cured tobacco plant or plant component of the inventiondesirably includes a recombinant nicotine demethylase gene, e.g., onecontaining the sequence of SEQ ID NO:1 or SEQ ID NO:3, or a fragmentthereof. Desirably, the expression of an endogenous nicotine demethylasegene in the cured tobacco plant or plant component is silenced.

Another aspect of the invention features a tobacco product containing acured tobacco plant or plant component that includes (i) reducedexpression of a nicotine demethylase or (ii) a nicotine demethylasehaving altered activity, and a reduced amount of a nitrosamine.Desirably, the tobacco product is smokeless tobacco, moist or dry snuff,a chewing tobacco, cigarette, cigar, cigarillo, pipe tobacco, or bidis.In particular, the tobacco product of this aspect of the invention maycontain dark tobacco, milled tobacco, or include a flavoring component.

The invention also features a method of making a tobacco product, e.g.,a smokeless tobacco product, containing (i) reduced expression of anicotine demethylase or (ii) a nicotine demethylase having altered(e.g., reduced) enzymatic activity, and a reduced amount of anitrosamine. This method involves providing a cured tobacco plant orplant component containing (i) a reduced level of nicotine demethylaseor (ii) a nicotine demethylase having an altered enzymatic activity anda reduced amount of a nitrosamine and preparing the tobacco product fromthe cured tobacco plant or plant component.

DEFINITIONS

“Enzymatic activity” is meant to include but is not limited todemethylation, hydroxylation, epoxidation, N-oxidation, sulfooxidation,N-, S-, and 0-dealkylations, desulfation, deamination, and reduction ofazo, nitro, N-oxide, and other such enzymatically reactive chemicalgroups. Altered enzymatic activity refers to a decrease in enzymaticactivity (for example, of a tobacco nicotine demethylase) by at least10-20%, preferably by at least 25-50%, and more preferably by at least55-95% or greater relative to the activity of a control enzyme (forexample, a wild-type tobacco plant nicotine demethylase). The activityof a tobacco nicotine demethylase may be assayed using methods standardin the art, such as by measuring the demethylation of radioactivenicotine by yeast-expressed microsomes, as described herein.

The term “nucleic acid” refers to a deoxyribonucleotide orribonucleotide polymer in either single- or double-stranded form, orsense or anti-sense, and unless otherwise limited, encompasses knownanalogues of natural nucleotides that hybridize to nucleic acids in amanner similar to naturally occurring nucleotides. Unless otherwiseindicated, a particular nucleic acid sequence includes the complementarysequence thereof. The terms “operably linked,” “in operablecombination,” and “in operable order” refer to functional linkagebetween a nucleic acid expression control sequence (such as a promoter,signal sequence, or array of transcription factor binding sites) and asecond nucleic acid sequence, wherein the expression control sequenceaffects transcription and/or translation of the nucleic acidcorresponding to the second sequence.

The term “recombinant” when used with reference to a cell indicates thatthe cell replicates a heterologous nucleic acid, expresses the nucleicacid or expresses a peptide, heterologous peptide, or protein encoded bya heterologous nucleic acid. Recombinant cells can express genes or genefragments in either the sense or antisense form or an RNA moleculecapable of inducing RNA interference (RNAi) that are not found withinthe native (non-recombinant) form of the cell. Recombinant cells canalso express genes that are found in the native form of the cell, butwherein the genes are modified and re-introduced into the cell byartificial means.

A “structural gene” is that portion of a gene comprising a DNA segmentencoding a protein, polypeptide or a portion thereof, and excluding, forexample, the 5′ sequence which drives the initiation of transcription orthe 3′UTR. The structural gene may alternatively encode anontranslatable product. The structural gene may be one which isnormally found in the cell or one which is not normally found in thecell or cellular location wherein it is introduced, in which case it istermed a “heterologous gene.” A heterologous gene may be derived inwhole or in part from any source known to the art, including a bacterialgenome or episome, eukaryotic, nuclear or plasmid DNA, cDNA, viral DNAor chemically synthesized DNA. A structural gene may contain one or moremodifications that could affect biological activity or itscharacteristics, the biological activity or the chemical structure ofthe expression product, the rate of expression or the manner ofexpression control. Such modifications include, but are not limited to,mutations, insertions, deletions and substitutions of one or morenucleotides.

The structural gene may constitute an uninterrupted coding sequence orit may include one or more introns, bounded by the appropriate splicejunctions. The structural gene may be translatable or non-translatable,including an antisense or an RNA molecule capable of inducing RNAinterference (RNAi). The structural gene may be a composite of segmentsderived from a plurality of sources and from a plurality of genesequences (naturally occurring or synthetic, where synthetic refers toDNA that is chemically synthesized).

An “exon” as used herein in reference to a nucleic acid sequence ismeant a portion of the nucleic acid sequence of a gene, where thenucleic acid sequence of the exon encodes at least one amino acid of thegene product. An exon is typically adjacent to a noncoding DNA segmentsuch as an intron. Desirably, an exon encodes a portion of the tobacconicotine demethylase amino acid sequence of SEQ ID NO:2, such as aminoacids 1-313 and/or 314-517 of the sequence of SEQ ID NO:2.

An “intron” as used herein in reference to a nucleic acid sequence ismeant a non-coding region of a gene that is flanked by coding regions.An intron is typically a noncoding region of a gene that is transcribedinto an RNA molecule but is then excised by RNA splicing duringproduction of the messenger RNA or other functional structural RNA.Desirably, an intron includes the sequence of SEQ ID NO:5, or a fragmentthereof.

A “3′UTR” as used herein in reference to a nucleic acid sequence ismeant a non-coding nucleic acid sequence proximal to a stop codon of anexon. Desirably, a 3′UTR includes the sequence of SEQ ID NO:7, or afragment thereof.

“Derived from” is used to mean taken, obtained, received, traced,replicated or descended from a source (chemical and/or biological). Aderivative may be produced by chemical or biological manipulation(including, but not limited to, substitution, addition, insertion,deletion, extraction, isolation, mutation and replication) of theoriginal source.

“Chemically synthesized,” as related to a sequence of DNA, means thatportions of the component nucleotides were assembled in vitro. Manualchemical synthesis of DNA may be accomplished using well-establishedprocedures (Caruthers, Methodology of DNA and RNA Sequencing, (1983),Weissman (ed.), Praeger Publishers, New York, Chapter 1); automatedchemical synthesis can be performed using one of a number ofcommercially available machines.

Optimal alignment of sequences for comparison may be conducted, forexample, by the local homology algorithm of Smith and Waterman, Adv.Appl. Math. 2:482 (1981), by the homology alignment algorithm ofNeedleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search forsimilarity method of Pearson and Lipman Proc. Natl. Acad. Sci. (U.S.A.)85: 2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or byinspection.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al.,1990) is available from several sources, including the National Centerfor Biological Information (NCBI, Bethesda, Md.) and on the internet,for use in connection with the sequence analysis programs blastp,blastn, blastx, tblastn, and tblastx. It can be accessed athttp://www.ncbi.nlm.nih.gov/BLAST/. A description of how to determinesequence identity using this program is available athttp://www.ncbi.nlm.nih.gov/BLAST/blast help.html.

The terms “substantial amino acid identity” or “substantial amino acidsequence identity” as applied to amino acid sequences and as used hereindenote a characteristic of a polypeptide, wherein the peptide comprisesa sequence that has at least 70 percent sequence identity, preferably 80percent amino acid sequence identity, more preferably 90 percent aminoacid sequence identity, and most preferably at least 99 to 100 percentsequence identity as compared to the protein sequence of SEQ ID NOS:2and/or 4. Desirably, for a nicotine demethylase, sequence comparison isdesirably compared for a region following the cytochrome p450 motif(GXRXCX(G/A); SEQ ID NO:29) to the stop codon of the translated peptide.

The terms “substantial nucleic acid identity” or “substantial nucleicacid sequence identity” as applied to nucleic acid sequences and as usedherein denote a characteristic of a polynucleotide sequence, wherein thepolynucleotide comprises a sequence that has at least 50 percent,preferably 60, 65, 70, or 75 percent sequence identity, more preferably81 or 91 percent nucleic acid sequence identity, and most preferably atleast 95, 99, or even 100 percent sequence identity as compared to SEQID NOS:1, 3, 5, 6, and/or 7. Desirably, for a nicotine demethylasenucleic acid sequence, comparison is desirably compared for a sequenceencoding a region following the cytochrome p450 motif (GXRXCX(G/A); SEQID NO:29) to the stop codon of the translated peptide.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other under stringent conditions.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Generally, stringent conditions are selected tobe about 5° C. to about 20° C., usually about 10° C. to about 15° C.,lower than the thermal melting point (Tm) for the specific sequence at adefined ionic strength and pH. The Tm is the temperature (under definedionic strength and pH) at which 50% of the target sequence hybridizes toa matched probe. Typically, stringent conditions will be those in whichthe salt concentration is about 0.02 molar at pH 7 and the temperatureis at least about 60° C. For instance in a standard Southernhybridization procedure, stringent conditions will include an initialwash in 6×SSC at 42° C. followed by one or more additional washes in0.2×SSC at a temperature of at least about 55° C., typically about 60°C., and often about 65° C.

Nucleotide sequences are also substantially identical for purposes ofthis invention when said nucleotide sequences encode polypeptides and/orproteins which are substantially identical. Thus, where one nucleic acidsequence encodes essentially the same polypeptide as a second nucleicacid sequence, the two nucleic acid sequences are substantiallyidentical even if they would not hybridize under stringent conditionsdue to degeneracy permitted by the genetic code (see, Darnell et al.(1990) Molecular Cell Biology, Second Edition Scientific American BooksW. H. Freeman and Company New York for an explanation of codondegeneracy and the genetic code). Protein purity or homogeneity can beindicated by a number of means well known in the art, such aspolyacrylamide gel electrophoresis of a protein sample, followed byvisualization upon staining. For certain purposes high resolution may beneeded and HPLC or a similar means for purification may be used.

By an antibody that “specifically binds” or “specifically recognizes” atobacco nicotine demethylase is meant an increased affinity of theantibody for a tobacco nicotine demethylase relative to an equal amountof any other protein. For example, an antibody that specifically bindsto a tobacco nicotine demethylase containing the amino acid sequence ofSEQ ID NO:2 desirably has an affinity for its antigen that is least2-fold, 5-fold, 10-fold, 30-fold, or 100-fold greater than for an equalamount of any other antigen, including related antigens. Binding of anantibody to an antigen, e.g., tobacco nicotine demethylase, may bedetermined by any number of standard methods in the art, e.g., Westernanalysis, ELISA, or co-immunoprecipitation.

As used herein, the term “vector” is used in reference to nucleic acidmolecules that transfer DNA segment(s) into a cell. A vector may act toreplicate DNA and may reproduce independently in a host cell. The term“vehicle” is sometimes used interchangeably with “vector.” The term“expression vector” as used herein refers to a recombinant DNA moleculecontaining a desired coding sequence and appropriate nucleic acidsequences necessary for the expression of the operably linked codingsequence in a particular host organism. Nucleic acid sequences necessaryfor expression in prokaryotes usually include a promoter, an operator(optional), and a ribosome binding site, often along with othersequences. Desirably, the promoter includes the sequence of SEQ ID NO:6,or a fragment thereof that drives transcription. Also desirable arepromoter sequences that have at least 50%, 60%, 75%, 80%, 90%, 95%, oreven 99% sequence identity to the sequence of SEQ ID NO:6 and that drivetranscription. Eucaryotic cells are known to utilize promoters,enhancers, and termination and polyadenylation signals, such as the3′UTR sequence of SEQ ID NO:7. In some instances, it has been observedthat plant expression vectors require the presence of plant derivedintrons, such as the intron having the sequence of SEQ ID NO:5, to havestable expression. As such, the sequence of SEQ ID NO:5, or any otherintron having an appropriate RNA splice junction may be used as furtherdescribed herein.

For the purpose of regenerating complete genetically engineered plantswith roots, a nucleic acid may be inserted into plant cells, forexample, by any technique such as in vivo inoculation or by any of theknown in vitro tissue culture techniques to produce transformed plantcells that can be regenerated into complete plants. Thus, for example,the insertion into plant cells may be by in vitro inoculation bypathogenic or non-pathogenic A. tumefaciens. Other such tissue culturetechniques may also be employed.

“Plant tissue,” “plant component” or “plant cell” includesdifferentiated and undifferentiated tissues of plants, including, butnot limited to, roots, shoots, leaves, pollen, seeds, tumor tissue andvarious forms of cells in culture, such as single cells, protoplasts,embryos and callus tissue. The plant tissue may be in planta or inorgan, tissue or cell culture.

“Plant cell” as used herein includes plant cells in planta and plantcells and protoplasts in culture. “cDNA” or “complementary DNA”generally refers to a single stranded DNA molecule with a nucleotidesequence that is complementary to an unprocessed RNA molecule containingan intron, or a processed mRNA lacking introns. cDNA is formed by theaction of the enzyme reverse transcriptase on an RNA template.

“Tobacco” as used herein includes flue-cured, Virginia, Burley, dark,oriental, and other types of plant within the genus Nicotiana. Seed ofthe genus Nicotiana is readily available commercially in the form ofNicotiana tabacum.

“Articles of manufacture” or “tobacco products” include products such asmoist and dry snuff, chewing tobaccos, cigarettes, cigars, cigarillos,pipe tobaccos, bidis, and similar tobacco-derived products.

By “gene silencing” is meant a decrease in the level of gene expression(for example, expression of a gene encoding a tobacco nicotinedemethylase) by at least 30-50%, preferably by at least 50-80%, and morepreferably by at least 80-95% or greater relative to the level in acontrol plant (for example, a wild-type tobacco plant). Reduction ofsuch expression levels may be accomplished by employing standard methodswhich are known in the art including, without limitation, RNAinterference, triple strand interference, ribozymes, homologousrecombination, virus-induced gene silencing, antisense andco-suppression technologies, expression of a dominant negative geneproduct, or through the generation of mutated genes using standardmutagenesis techniques, such as those described herein. Levels of atobacco nicotine demethylase polypeptide or transcript, or both, aremonitored according to any standard technique including, but not limitedto, Northern blotting, RNase protection, or immunoblotting.

By a “tobacco nicotine demethylase” or “nicotine demethylase” as usedherein, is meant a polypeptide that is substantially identical to thesequence of SEQ ID NO:2. Desirably, a tobacco nicotine demethylase iscapable of converting nicotine (C₁₀H₁₂N₂, also referred to as341-Methyl-2-pyrrolidinyl)pyridine) to nornicotine (C₉H₁₂N₂). Theactivity of a tobacco nicotine demethylase may be assayed using methodsstandard in the art, such as by measuring the demethylation ofradioactive nicotine by yeast-expressed microsomes, as described herein.

By a “fragment” or “portion” of a tobacco nicotine demethylase aminoacid sequence is meant at least e.g., 20, 15, 30, 50, 75, 100, 250, 300,400, or 500 contiguous amino acids of the amino acid sequence of SEQ IDNO:2. Exemplary desirable fragments are amino acids 1-313 of thesequence of SEQ ID NO:2 and amino acids 314-517 of the sequence of SEQID NO:2. In addition, with respect to a fragment or portion of a tobacconicotine demethylase nucleic acid sequence, desirable fragments includeat least 100, 250, 500, 750, 1000, or 1500 contiguous nucleic acids ofthe nucleic acid sequence of SEQ ID NO:1. Exemplary desirable fragmentsare nucleic acids 1-2009, 2010-2949, 2950-3946, 3947-4562, 4563-6347,and 4731-6347 of the sequence of SEQ ID NO:1. Other desirable fragmentsinclude nucleic acids 1-100, 101-250, 251-500, 501-750, and 751-998 ofSEQ ID NO:5, nucleic acids 1-398, 1-1400, 1401-2009, 1840-2009,1940-2009, 399-1240, and 1241-2009 of SEQ ID NO:6, and nucleic acids1-100, 101-250, 251-500, 501-750, 751-1000, 1001-1250, 1251-1500, and1501-1786 of SEQ ID NO:7.

By a “substantially pure polypeptide” is meant a tobacco nicotinedemethylase that has been separated from most components which naturallyaccompany it; however, other proteins found in the microsomal fractionassociated with a preparation having a nicotine demethylase activity ofat least 8.3 pKat/mg protein, 9 pKat/mg protein, 9.5 pKat/mg protein, 10pKat/mg protein, 10.5 pKat/mg, or 10.8 pKat/mg protein are alsoconsidered to be a substantially pure polypeptide. Typically, thepolypeptide is substantially pure when it is at least 60%, by weight,free from the proteins and naturally-occurring organic molecules withwhich it is naturally associated. Preferably, the preparation is atleast 75%, more preferably at least 90%, and most preferably at least99%, by weight, a tobacco nicotine demethylase. A substantially puretobacco nicotine demethylase may be obtained, for example, by extractionfrom a natural source (for example, a tobacco plant cell); by expressionof a recombinant nucleic acid encoding a tobacco nicotine demethylase;or by chemically synthesizing the protein. Purity can be measured by anyappropriate method, for example, column chromatography, polyacrylamidegel electrophoresis, or by HPLC analysis.

By “isolated nucleic acid molecule” is meant a nucleic acid sequencefree from the nucleic acid sequences that naturally flank the sequenceof the nucleic acid molecule in the genome of an organism.

By “transformed cell” is meant a cell into which (or into an ancestor ofwhich) has been introduced, by means of recombinant DNA techniques, aDNA molecule, for example, a DNA molecule encoding a tobacco nicotinedemethylase.

As provided herein, the terms “cytochrome p450” and “p450” are usedinterchangeably.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of the genomic structure of the tobacconicotine demethylase gene.

FIGS. 2-1 to 2-3 is the genomic tobacco nicotine demethylase nucleicacid sequence (SEQ ID NO:1) and its translation product (SEQ ID NO:2).

FIG. 3 is the nucleic acid sequence of the tobacco nicotine demethylasecoding region (SEQ ID NO:3) (also referred to as D121-AA8) and itstranslation product (SEQ ID NO:4).

FIG. 4 is the nucleic acid sequence of an intron (SEQ ID NO:5) presentin the tobacco nicotine demethylase genomic sequence.

FIG. 5 is the nucleic acid sequence of the tobacco nicotine demethylasepromoter region (SEQ ID NO:6).

FIG. 6 is the nucleic acid sequence of the 3′UTR of the tobacco nicotinedemethylase gene (SEQ ID NO:7).

DETAILED DESCRIPTION

Identifying a Genomic Clone Encoding a Tobacco Nicotine Demethylase

In accordance with the present invention, RNA was extracted fromNicotiana tissue of converter and non-converter Nicotiana lines. Theextracted RNA was then used to create cDNA. Nucleic acid sequences ofthe present invention were then generated using two strategies.

In the first strategy, the poly A enriched RNA was extracted from planttissue and cDNA was made by reverse transcription PCR. The single strandcDNA was then used to create p450 specific PCR populations usingdegenerate primers plus a oligo d(T) reverse primer. The primer designwas based on the highly conserved motifs of other plant cytochrome p450gene sequences. Examples of specific degenerate primers are set forth inFIG. 1 of the US 2004/0103449 A1 and US 2004/0111759 A1 patentapplication publications, which are hereby incorporated by reference.The sequence of fragments from plasmids containing appropriate sizeinserts was further analyzed. These size inserts typically ranged fromabout 300 to about 800 nucleotides depending on which primers were used.

In a second strategy, a cDNA library was initially constructed. The cDNAin the plasmids was used to create p450 specific PCR populations usingdegenerate primers plus T7 primer on plasmid as reverse primer. As inthe first strategy, the sequence of fragments from plasmids containingappropriate size inserts was further analyzed.

Nicotiana plant lines known to produce high levels of nornicotine(converter) and plant lines having low levels of nornicotine may be usedas starting materials. Leaves can then be removed from plants andtreated with ethylene to activate p450 enzymatic activities definedherein. Total RNA is extracted using techniques known in the art. cDNAfragments can then be generated using PCR(RT-PCR) with the oligo d(T)primer. The cDNA library can then be constructed as more fully describedin examples herein.

The conserved region of p450 type enzymes was used as a template fordegenerate primers. Using degenerate primers, p450 specific bands wereamplified by PCR. Bands indicative for p450-like enzymes were identifiedby DNA sequencing. PCR fragments were characterized using BLAST search,alignment or other tools to identify appropriate candidates.

Sequence information from identified fragments was used to develop PCRprimers. These primers in combination with plasmid primers in cDNAlibrary were used to clone full-length p450 genes. Large-scale Southernreverse analysis was conducted to examine the differential expressionfor all fragment clones obtained and in some cases full-length clones.In this aspect of the invention, these large-scale reverse Southernassays can be conducted using labeled total cDNAs from different tissuesas a probe to hybridize with cloned DNA fragments in order to screen allcloned inserts. Nonradioactive and radioactive (P32) Northern blottingassays were also used to characterize cloned p450 fragments andfull-length clones.

Peptide specific antibodies were made by deriving their amino acidsequence and selecting peptide regions that were antigenic and uniquerelative to other clones. Rabbit antibodies were made to syntheticpeptides conjugated to a carrier protein. Western blotting analyses orother immunological methods were performed on plant tissue using theseantibodies. In addition, peptide specific antibodies were made forseveral full-length clones by deriving their amino acid sequence andselecting peptide regions that were potentially antigenic and wereunique relative to other clones. Rabbit antibodies were made tosynthetic peptides conjugated to a carrier protein. Western blottinganalyses were performed using these antibodies.

Downregulating Tobacco Nicotine Demethylase

Plants having decreased expression of a tobacco nicotine demethylase aregenerated according to standard gene silencing methods. (For reviews,see Arndt and Rank, Genome 40:785-797, 1997; Turner and Schuch, Journalof Chemical Technology and Biotechnology 75:869-882, 2000; and Klink andWolniak, Journal of Plant Growth Regulation 19(4):371-384, 2000.) Inparticular, the tobacco nicotine demethylase gene promoter (e.g., SEQ IDNO:6), structural gene (SEQ ID NO:3), intron (SEQ ID NO:5), or 3′ UTR(SEQ ID NO:7) or the entire genomic clone (SEQ ID NO:1) can be used toalter tobacco phenotypes or tobacco metabolites, for example,nornicotine in any Nicotiana species. Decreased expression of a tobacconicotine demethylase gene may be achieved using, for example, RNAinterference (RNAi) (Smith et al., Nature 407:319-320, 2000; Fire etal., Nature 391:306-311, 1998; Waterhouse et al., PNAS 95:13959-13964,1998; Stalberg et al., Plant Molecular Biology 23:671-683, 1993;Brignetti et al., EMBO J. 17:6739-6746, 1998; Allen et al., NatureBiotechnology 22: 1559-1566, 2004); virus-induced gene silencing(“VIGS”) (Baulcombe, Current Opinions in Plant Biology, 2:109-113, 1999;Cogoni and Macino, Genes Dev 10: 638-643, 2000; Ngelbrecht et al., PNAS91:10502-10506, 1994); silencing the target gene by transferring a plantendogenous gene in the sense orientation (Jorgensen et al., Plant MolBiol 31: 957-973, 1996); expression of antisense gene; homologousrecombination (Ohl et al., Homologous Recombination and Gene Silencingin Plants (Kluwer, Dordrecht, The Netherlands, 1994); Cre/lox systems(Qin et al., PNAS 91: 1706-1710, 1994; Koshinsky et al., The PlantJournal 23: 715-722, 2000; Chou, et al., Plant and Animal Genome VIIConference Abstracts. San Diego, Calif., 17-21 Jan. 1999); gene trappingand T-DNA tagging (Burns et al., Genes Dev. 8: 1087-1105, 1994;Spradling, et al., PNAS 92:10824-10830, 1995; Skarnes et al.,Bio/Technology 8, 827-831, 1990; Sundaresan, et al., Genes Dev. 9:1797-1810, 1995); and any of the other possible gene silencing systemsthat are available in the science areas that result in thedownregulation of expression of a tobacco nicotine demethylase or in areduction in its enzymatic activity. Exemplary methods are described inmore detail below.

RNA Interference

RNA interference (“RNAi”) is a generally applicable process for inducingpotent and specific post-translational gene silencing in many organismsincluding plants (see, e.g., Bosher et al., Nat. Cell Biol. 2:E31-36,2000; and Tavernarakis et al., Nat. Genetics 24:180-183, 2000). RNAiinvolves introduction of RNA with partial or fully double-strandedcharacter into the cell or into the extracellular environment.Inhibition is specific in that a nucleotide sequence from a portion ofthe target gene (e.g., a tobacco nicotine demethylase) is chosen toproduce inhibitory. RNA. The chosen portion generally encompasses exonsof the target gene, but the chosen portion may also include untranslatedregions (UTRs), as well as introns (e.g., the sequences of SEQ ID NO:5or 7).

For example, to construct transformation vectors that produce RNAscapable of duplex formation, two tobacco nicotine demethylase nucleicacid sequences, one in the sense and the other in the antisenseorientation, may be operably linked, and placed under the control of astrong viral promoter, such as CaMV 35S or the promoter isolated fromcassava brown streak virus (CBSV). However, use of the endogenouspromoter, such as the tobacco nicotine demethylase promoter having thesequence of SEQ ID NO:6, or a fragment thereof that drivestranscription, may also be desirable. The length of the tobacco nicotinedemethylase nucleic acid sequences included in such a construct isdesirably at least 25 nucleotides, but may encompass a sequence thatincludes up to the full-length tobacco nicotine demethylase gene.

Constructs that produce RNAs capable of duplex formation may beintroduced into the genome of a plant, such as a tobacco plant, byAgrobacterium-mediated transformation (Chuang et al., Proc. Natl. Acad.Sci. USA 97:4985-4990, 2000), causing specific and heritable geneticinterference in a tobacco nicotine demethylase. The double-stranded RNAmay also be directly introduced into the cell (i.e., intracellularly) orintroduced extracellularly, for example, by bathing a seed, seedling, orplant in a solution containing the double-stranded RNA.

Depending on the dose of double-stranded RNA material delivered, theRNAi may provide partial or complete loss of function for the targetgene. A reduction or loss of gene expression in at least 99% of targetedcells may be obtained. In general, lower doses of injected material andlonger times after administration of dsRNA result in inhibition in asmaller fraction of cells.

The RNA used in RNAi may comprise one or more strands of polymerizedribonucleotide; it may include modifications to either thephosphate-sugar backbone or the nucleoside. The double-strandedstructure may be formed by a single self-complementary RNA strand or bytwo complementary RNA strands and RNA duplex formation may be initiatedeither inside or outside the cell. The RNA may be introduced in anamount which allows delivery of at least one copy per cell. However,higher doses (e.g., at least 5, 10, 100, 500 or 1000 copies per cell) ofdouble-stranded material may yield more effective inhibition. Inhibitionis sequence-specific in that nucleotide sequences corresponding to theduplex region of the RNA are targeted for genetic inhibition. RNAcontaining a nucleotide sequences identical to a portion of the targetgene is preferred for inhibition. RNA sequences with insertions,deletions, and single point mutations relative to the target sequencemay also be effective for inhibition. Thus, sequence identity may beoptimized by alignment algorithms known in the art and calculating thepercent difference between the nucleotide sequences. Alternatively, theduplex region of the RNA may be defined functionally as a nucleotidesequence that is capable of hybridizing with a portion of the targetgene transcript.

In addition, the RNA used for RNAi may be synthesized either in vivo orin vitro. For example, endogenous RNA polymerase in the cell may mediatetranscription in vivo, or cloned RNA polymerase can be used fortranscription in vivo or in vitro. For transcription from a transgene invivo or an expression construct, a regulatory region may be used totranscribe the RNA strand (or strands).

Triple Strand Interference

Endogenous tobacco nicotine demethylase gene expression may also bedownregulated by targeting deoxyribonucleotide sequences complementaryto the regulatory region of a tobacco nicotine demethylase gene (e.g.,promoter or enhancer regions) to form triple helical structures thatprevent transcription of the tobacco nicotine demethylase gene in targetcells. (See generally, Helene, Anticancer Drug Des. 6:569-584, 1991;Helene et al., Ann. N.Y. Acad. Sci. 660:27-36, 1992; and Maher,Bioassays 14:807-815, 1992.)

Nucleic acid molecules used in triple helix formation for the inhibitionof transcription are preferably single stranded and composed ofdeoxyribonucleotides. The base composition of these oligonucleotidesshould promote triple helix formation via Hoogsteen base pairing rules,which generally require sizable stretches of either purines orpyrimidines to be present on one strand of a duplex. Nucleotidesequences may be pyrimidine-based, which will result in TAT and CGCtriplets across the three associated strands of the resulting triplehelix. The pyrimidine-rich molecules provide base complementarity to apurine-rich region of a single strand of the duplex in a parallelorientation to that strand. In addition, nucleic acid molecules may bechosen that are purine-rich, for example, containing a stretch of Gresidues. These molecules will form a triple helix with a DNA duplexthat is rich in GC pairs, in which the majority of the purine residuesare located on a single strand of the targeted duplex, resulting in CGCtriplets across the three strands in the triplex.

Alternatively, the potential sequences that can be targeted for triplehelix formation may be increased by creating a “switchback” nucleic acidmolecule. Switchback molecules are synthesized in an alternating5′-3′,3′-5′ manner, such that they base pair with first one strand of aduplex and then the other, eliminating the necessity for a sizablestretch of either purines or pyrimidines to be present on one strand ofa duplex.

Ribozymes

Ribozymes are RNA molecules that act as enzymes and can be engineered tocleave other RNA molecules. A ribozyme may be designed to specificallypair with virtually any target RNA and cleave the phosphodiesterbackbone at a specific location, thereby functionally inactivating thetarget RNA. The ribozyme itself is not consumed in this process and canact catalytically to cleave multiple copies of mRNA target molecules.Accordingly, ribozymes may also be used as a means to downregulateexpression of a tobacco nicotine demethylase. The design and use oftarget RNA-specific ribozymes is described in Haseloff et al. (Nature334:585-591, 1988). Preferably, the ribozyme includes at least about 20continuous nucleotides complementary to the target sequence (e.g., atobacco nicotine demethylase) on each side of the active site of theribozyme.

In addition, ribozyme sequences may also be included within an antisenseRNA to confer RNA-cleaving activity upon the antisense RNA and, thereby,increasing the effectiveness of the antisense construct.

Homologous Recombination

Gene replacement technology is another desirable method fordownregulating expression of a given gene, e.g., a tobacco nicotinedemethylase. Gene replacement technology is based upon homologousrecombination (see, Schnable et al., Curr. Opinions Plant Biol.1:123-129, 1998). The nucleic acid sequence of the enzyme of interestsuch as a tobacco nicotine demethylase can be manipulated by mutagenesis(e.g., insertions, deletions, duplications or replacements) to decreaseenzymatic function. The altered sequence can then be introduced into thegenome to replace the existing, e.g., wild-type, gene via homologousrecombination (Puchta et al., Proc. Natl. Acad. Sci. USA 93:5055-5060,1996; and Kempin et al., Nature 389: 802-803, 1997).

Co-Suppression

A further desirable method of silencing gene expression isco-suppression (also referred to as sense suppression). This technique,which involves introduction of a nucleic acid configured in the senseorientation, has been shown to effectively block the transcription oftarget genes (see, for example, Napoli et al., Plant Cell, 2:279-289,1990 and Jorgensen et al., U.S. Pat. No. 5,034,323).

Generally, sense suppression involves transcription of the introducedsequence. However, co-suppression may also occur where the introducedsequence contains no coding sequence per se, but only intron (e.g., thesequence of SEQ ID NO:5) or untranslated sequences such as the sequenceof SEQ ID NO:7 or other such sequences substantially identical tosequences present in the primary transcript of the endogenous gene to berepressed. The introduced sequence generally will be substantiallyidentical to the endogenous gene targeted for repression. Such identityis typically greater than about 50%, but higher identities (for example,80% or even 95%) are preferred because they result in more effectiverepression. The effect of co-suppression may also be applied to otherproteins within a similar family of genes exhibiting homology orsubstantial homology. Segments from a gene from one plant can be useddirectly, for example, to inhibit expression of homologous genes indifferent plant species.

In sense suppression, the introduced sequence, requiring less thanabsolute identity, need not be full length, relative to either theprimary transcription product or to fully processed mRNA. A higherdegree of sequence identity in a shorter than full-length sequencecompensates for a longer sequence of lesser identity. Furthermore, theintroduced sequence need not have the same intron or exon pattern, andidentity of non-coding segments may be equally effective. Sequences ofat least 50 base pairs are preferred, with introduced sequences ofgreater length being more preferred (see, for example, those methodsdescribed by Jorgensen et al., U.S. Pat. No. 5,034,323).

Antisense Suppression

In antisense technology, a nucleic acid segment from the desired plantgene, such as that found in SEQ ID NOS:1, 3, 5, 6, or 7, is cloned andoperably linked to an expression control region such that the antisensestrand of RNA is synthesized. The construct is then transformed intoplants and the antisense strand of RNA is produced. In plant cells, ithas been shown that antisense RNA inhibits gene expression.

The nucleic acid segment to be introduced in antisense suppression isgenerally substantially identical to at least a portion of theendogenous gene or genes to be repressed, but need not be identical. Thenucleic acid sequences of the tobacco nicotine demethylase disclosedherein may be included in vectors designed such that the inhibitoryeffect applies to other proteins within a family of genes exhibitinghomology or substantial homology to the target gene. Segments from agene from one plant can be used, for example, directly to inhibitexpression of homologous genes in different tobacco varieties.

The introduced sequence also need not be full length relative to eitherthe primary transcription product or to fully processed mRNA. Generally,higher homology can be used to compensate for the use of a shortersequence. Moreover, the introduced sequence need not have the sameintron or exon pattern, and homology of non-coding segments will beequally effective. In general, such an antisense sequence will usuallybe at least 15 base pairs, preferably about 15-200 base pairs, and morepreferably 200-2,000 base pairs in length or greater. The antisensesequence may be complementary to all or a portion of the gene to besuppressed (for example, a tobacco nicotine demethylase promoter (SEQ IDNO:6), exon, intron (SEQ ID NO:5), or UTR (SEQ ID NO:7), and, asappreciated by those skilled in the art, the particular site or sites towhich the antisense sequence binds as well as the length of theantisense sequence will vary, depending upon the degree of inhibitiondesired and the uniqueness of the antisense sequence. A transcriptionalconstruct expressing a plant negative regulator antisense nucleotidesequence includes, in the direction of transcription, a promoter, thesequence coding for the antisense RNA on the sense strand, and atranscriptional termination region. Antisense sequences may beconstructed and expressed as described, for example, in van der Krol etal. (Gene 72: 45-50, 1988); Rodermel et al. (Cell 55: 673-681, 1988);Mol et al. (FEBS Lett. 268: 427-430, 1990); Weigel and Nilsson (Nature377: 495-500, 1995); Cheung et al., (Cell 82: 383-393, 1995); andShewmaker et al. (U.S. Pat. No. 5,107,065).

Dominant Negatives

Transgenic plants expressing a transgene encoding a dominant negativegene product of a tobacco nicotine demethylase may be assayed inartificial environments or in the field to demonstrate that thetransgene confers downregulates a tobacco nicotine demethylase in thetransgenic plant. Dominant negative transgenes are constructed accordingto methods known in the art. Typically, a dominant negative gene encodesa mutant negative regulator polypeptide of a tobacco nicotinedemethylase which, when overexpressed, disrupts the activity of thewild-type enzyme.

Mutants

Plants having decreased expression or enzymatic activity of a tobacconicotine demethylase may also be generated using standard mutagenesismethodologies. Such mutagenesis methods include, without limitation,treatment of seeds with ethyl methylsulfate (Hildering and Verkerk, In,The use of induced mutations in plant breeding. Pergamon press, pp317-320, 1965) or UV-irradiation, X-rays, and fast neutron irradiation(see, for example, Verkerk, Neth. J. Agric. Sci. 19:197-203, 1971; andPoehlman, Breeding Field Crops, Van Nostrand Reinhold, New York(3.sup.rd ed), 1987), use of transposons (Fedoroff et al., 1984; U.S.Pat. No. 4,732,856 and U.S. Pat. No. 5,013,658), as well as T-DNAinsertion methodologies (Hoekema et al., 1983; U.S. Pat. No. 5,149,645).The types of mutations that may be present in a tobacco nicotinedemethylase gene include, for example, point mutations, deletions,insertions, duplications, and inversions. Such mutations desirably arepresent in the coding region of a tobacco nicotine demethylase gene;however mutations in the promoter region, and intron, or an untranslatedregion of a tobacco nicotine demethylase gene may also be desirable.

For instance, T-DNA insertional mutagenesis may be used to generateinsertional mutations in a tobacco nicotine demethylase gene todownregulate the expression of the gene. Theoretically, about 100,000independent T-DNA insertions are required for a 95% probability ofgetting an insertion in any given gene (McKinnet, Plant J. 8: 613-622,1995; and Forsthoefel et al., Aust. J. Plant Physiol. 19:353-366, 1992).T-DNA tagged lines of plants may be screened using polymerase chainreaction (PCR) analysis. For example, a primer can be designed for oneend of the T-DNA and another primer can be designed for the gene ofinterest and both primers can be used in the PCR analysis. If no PCRproduct is obtained, then there is no insertion in the gene of interest.In contrast, if a PCR product is obtained, then there is an insertion inthe gene of interest.

Expression of a mutated tobacco nicotine demethylase may be evaluatedaccording to standard procedures (for example, those described herein)and, optionally, may be compared to expression of the non-mutatedenzyme. When compared to non-mutated plants, mutated plants havingdecreased expression of a gene encoding a tobacco nicotine demethylaseare desirable embodiments of the present invention.

Plant Promoters

An example of a useful plant promoter according to the invention is thenicotine demethylase promoter having the sequence of SEQ ID NO:6, or afragment thereof that drives transcription. Another desirable promoteris a caulimovirus promoter, for instance, a cauliflower mosaic virus(CaMV) promoter or the cassava vein mosaic virus (CsVMV) promoter. Thesepromoters confer high levels of expression in most plant tissues, andthe activity of these promoters is not dependent on virally encodedproteins. CaMV is a source for both the 35S and 19S promoters. Examplesof plant expression constructs using these promoters are known in theart. In most tissues of transgenic plants, the CaMV 35S promoter is astrong promoter. The CaMV promoter is also highly active in monocots.Moreover, activity of this promoter can be further increased (i.e.,between 2-10 fold) by duplication of the CaMV 35S promoter.

Other useful plant promoters include, without limitation, the nopalinesynthase (NOS) promoter, the octopine synthase promoter, figwort mosiacvirus (FMV) promoter, the rice actin promoter, and the ubiquitinpromoter system.

Exemplary monocot promoters include, without limitation, commelinayellow mottle virus promoter, sugar cane badna virus promoter, ricetungro bacilliform virus promoter, maize streak virus element, and wheatdwarf virus promoter.

For certain applications, it may be desirable to produce a tobacconicotine demethylase, such as a dominant negative mutant tobacconicotine demethylase, in an appropriate tissue, at an appropriate level,or at an appropriate developmental time. For this purpose, there areassortments of gene promoters, each with its own distinctcharacteristics embodied in its regulatory sequences, shown to beregulated in response to inducible signals such as the environment,hormones, and/or developmental cues. These include, without limitation,gene promoters that are responsible for heat-regulated gene expression,light-regulated gene expression (for example, the pea rbcS-3A; the maizerbcS promoter; the chlorophyll a/b-binding protein gene found in pea; orthe Arabssu promoter), hormone-regulated gene expression (for example,the abscisic acid (ABA) responsive sequences from the Em gene of wheat;the ABA-inducible HVA1 and HVA22, and rd29A promoters of barley andArabidopsis; and wound-induced gene expression (for example, of wunI),organ-specific gene expression (for example, of the tuber-specificstorage protein gene; the 23-kDa zein gene from maize described by; orthe French bean β-phaseolin gene), or pathogen-inducible promoters (forexample, the PR-1, prp-1, or (β-1,3 glucanase promoters, thefungal-inducible wirla promoter of wheat, and the nematode induciblepromoters, TobRB7-5A and Hmg-1, of tobacco and parsley, respectively).

Plant Expression Vectors

Typically, plant expression vectors include (1) a cloned plant gene(e.g., a tobacco nicotine demethylase gene) under the transcriptionalcontrol of 5′ and 3′ regulatory sequences (e.g., the tobacco nicotinedemethyase promoter (SEQ ID NO:6) and 3′UTR regions (SEQ ID NO:7)) and(2) a dominant selectable marker. Such plant expression vectors may alsocontain, if desired, a promoter regulatory region (for example, oneconferring inducible or constitutive, pathogen- or wound-induced,environmentally- or developmentally-regulated, or cell- ortissue-specific expression), a transcription initiation start site, aribosome binding site, an RNA processing signal, a transcriptiontermination site, and/or a polyadenylation signal.

Plant expression vectors may also optionally include RNA processingsignals, e.g, introns, which have been shown to be important forefficient RNA synthesis and accumulation. The location of the RNA splicesequences can dramatically influence the level of transgene expressionin plants. In view of this fact, an intron may be positioned upstream ordownstream of a tobacco nicotine demethylase coding sequence in thetransgene to alter levels of gene expression.

In addition to the aforementioned 5′ regulatory control sequences, theexpression vectors may also include regulatory control regions which aregenerally present in the 3′ regions of plant genes. For example, the 3′terminator region (e.g., the sequence of SEQ ID NO:7) may be included inthe expression vector to increase stability of the mRNA. One suchterminator region may be derived from the PI-II terminator region ofpotato. In addition, other commonly used terminators are derived fromthe octopine or nopaline synthase signals.

The plant expression vector also typically contains a dominantselectable marker gene used to identify those cells that have becometransformed. Useful selectable genes for plant systems include theaminoglycoside phosphotransferase gene of transposon Tn5 (Aph II), genesencoding antibiotic resistance genes, for example, those encodingresistance to hygromycin, kanamycin, bleomycin, neomycin, G418,streptomycin, or spectinomycin. Genes required for photosynthesis mayalso be used as selectable markers in photosynthetic-deficient strains.Finally, genes encoding herbicide resistance may be used as selectablemarkers; useful herbicide resistance genes include the bar gene encodingthe enzyme phosphinothricin acetyltransferase and conferring resistanceto the broad-spectrum herbicide Basta® (Bayer Cropscience DeutschlandGmbH, Langenfeld, Germany). Other selectable markers include genes thatprovide resistance to other such herbicides such as glyphosate and thelike, and imidazolinones, sulfonylureas, triazolopyrimidine herbicides,such as chlorosulfuron, bromoxynil, dalapon, and the like. Furthermore,genes encoding dihydrofolate reductase may be used in combination withmolecules such as methotrexate.

Efficient use of selectable markers is facilitated by a determination ofthe susceptibility of a plant cell to a particular selectable agent anda determination of the concentration of this agent which effectivelykills most, if not all, of the transformed cells. Some usefulconcentrations of antibiotics for tobacco transformation include, forexample, 20-100 μg/ml (kanamycin), 20-50 μg/ml (hygromycin), or 5-10μg/ml (bleomycin). A useful strategy for selection of transformants forherbicide resistance is described, for example, by Vasil (Cell Cultureand Somatic Cell Genetics of Plants, Vol I, II, III LaboratoryProcedures and Their Applications Academic Press, New York, 1984).

In addition to a selectable marker, it may be desirable to use areporter gene. In some instances a reporter gene may be used without aselectable marker. Reporter genes are genes which are typically notpresent or expressed in the recipient organism or tissue. The reportergene typically encodes for a protein which provide for some phenotypicchange or enzymatic property. Examples of such genes are provided inWeising et al. (Ann. Rev. Genetics 22:421, 1988), which is incorporatedherein by reference. Preferred reporter genes include without limitationglucuronidase (GUS) gene and GFP genes.

Upon construction of the plant expression vector, several standardmethods are available for introduction of the vector into a plant host,thereby generating a transgenic plant. These methods include (1)Agrobacterium-mediated transformation (A. tumefaciens or A. rhizogenes)(see, for example, Lichtenstein and Fuller In: Genetic Engineering, vol6, P W J Rigby, ed, London, Academic Press, 1987; and Lichtenstein, C.P., and Draper, J., In: DNA Cloning, Vol II, D. M. Glover, ed, Oxford,IRI Press, 1985; U.S. Pat. Nos. 4,693,976, 4,762,785, 4,940,838,5,004,863, 5,104,310, 5,149,645, 5,159,135, 5,177,010, 5,231,019,5,463,174, 5,469,976, and 5,464,763; and European Patent ApplicationNumbers 0131624B1, 0159418B1, 0120516, 0176112, 0116718, 0267159,0290799, 0292435, 0320500, 0604622, and 0627752), (2) the particledelivery system (see, for example, U.S. Pat. Nos. 4,945,050 and5,141,131), (3) microinjection protocols, (4) polyethylene glycol (PEG)procedures, (5) liposome-mediated DNA uptake, (6) electroporationprotocols (see, for example, WO 87/06614, WO 92/09696, and WO 93/21335;and U.S. Pat. Nos. 5,384,253 and 5,472,869, (7) the vortexing method, or(8) the so-called whiskers methodology (see, for example, Coffee et al.,U.S. Pat. Nos. 5,302,523 and 5,464,765). The type of plant tissue thatmay be transformed with an expression vector includes embryonic tissue,callus tissue type I and II, hypocotyls, meristem, and the like.

Once introduced into the plant tissue, the expression of the structuralgene may be assayed by any means known to the art, and expression may bemeasured as mRNA transcribed, protein synthesized, or the amount of genesilencing that occurs as determined by metabolite monitoring viachemical analysis of secondary alkaloids in tobacco (as describedherein; see also U.S. Pat. No. 5,583,021 which is hereby incorporated byreference). Techniques are known for the in vitro culture of planttissue, and in a number of cases, for regeneration into whole plants(see, e.g., U.S. Pat. Nos. 5,595,733 and 5,766,900). Procedures fortransferring the introduced expression complex to commercially usefulcultivars are known to those skilled in the art.

Once plant cells expressing the desired level of nicotine demethylase(or nornicotine or NNN or both) are obtained, plant tissues and wholeplants can be regenerated therefrom using methods and techniqueswell-known in the art. The regenerated plants are then reproduced byconventional means and the introduced genes can be transferred to otherstrains and cultivars by conventional plant breeding techniques.

Transgenic tobacco plants may incorporate a nucleic acid of any portionof the genomic gene in different orientations for eitherdown-regulation, for example, antisense orientation or in a form toinduce RNAi, or over-expression, for example, sense orientation.Over-expression of the nucleic acid sequence that encodes the entire ora functional part of an amino acid sequence of a full-length tobacconicotine demethylase gene is desirable for increasing the expression ofnicotine demethylase within Nicotiana lines.

Determination of Transcriptional or Translational Levels of a TobaccoNicotine Demthylase

Tobacco nicotine demethylase expression may be measured, for example, bystandard Northern blot analysis (Ausubel et al., Current Protocols inMolecular Biology, John Wiley & Sons, New York, N.Y., (2001), andSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, N.Y., (1989)) using a tobacco nicotine demethylase(or cDNA fragment) as a hybridization probe. Determination of RNAexpression levels may also be aided by reverse transcription PCR(rtPCR), including quantitative rtPCR (see, e.g., Kawasaki et al., inPCR Technology: Principles and Applications of DNA Amplification (H. A.Erlich, Ed.) Stockton Press (1989); Wang et al. in PCR Protocols: AGuide to Methods and Applications (M. A. Innis, et al., Eds.) AcademicPress (1990); and Freeman et al., Biotechniques 26:112-122 and 124-125,1999). Additional well-known techniques for determining expression of atobacco nicotine demethylase gene include in situ hybridization, andfluorescent in situ hybridization (see, e.g., Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley & Sons, New York, N.Y.,(2001)). The above standard techniques are also useful to compare theexpression level between plants, for example, between a plant having amutation in a tobacco nicotine demethylase gene and a control plant.

If desired, expression of a tobacco nicotine demethylase gene may bemeasured at the level of tobacco nicotine demethylase protein productionusing the same general approach and standard protein analysis techniquesincluding Bradford assays, spectrophotometric assays, and immunologicaldetection techniques, such as Western blotting or immunoprecipitationwith a tobacco nicotine demethylase-specific antibody (Ausubel et al.,Current Protocols in Molecular Biology, John Wiley & Sons, New York,N.Y., (2001), and Sambrook et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory, N.Y., (1989)).

Identification of Modulators of a Tobacco Nicotine Demethylase

Isolation of a tobacco nicotine demethylase cDNA also facilitates theidentification of molecules that increase or decrease expression atobacco nicotine demethylase. According to one approach, candidatemolecules are added at varying concentrations to a culture medium ofcells (for example, prokaryotic cells such as E. coli or eukaryoticcells such as yeast, mammalian, insect, or plant cells) expressing atobacco nicotine demethylase mRNA. Tobacco nicotine demethylaseexpression is then measured in the presence and absence of a candidatemolecule using standard methods such as those set forth herein.

Candidate modulators may be purified (or substantially purified)molecules or may be one component of a mixture of compounds. In a mixedcompound assay, tobacco nicotine demethylase expression is testedagainst progressively smaller subsets of the candidate compound pool(for example, produced by standard purification techniques, for example,HPLC) until a single compound or minimal compound mixture isdemonstrated to alter tobacco nicotine demethylase gene expression. Amolecule that promotes a decrease in tobacco nicotine demethylaseexpression is considered particularly useful in the invention.Modulators found to be effective at the level of tobacco nicotinedemethylase expression or activity may be confirmed as useful in planta.

For agricultural uses, the molecules, compounds, or agents identifiedusing the methods disclosed herein may be used as chemicals applied assprays or dusts on the foliage of plants. The molecules, compounds, oragents may also be applied to plants in combination with anothermolecule which affords some benefit to the plant.

Uses of the Nicotine Demethylase Gene Promoter and Non-TranslatedRegions

The promoter region of the nicotine demethylase gene described herein isethylene inducible or related to plant senescence. Accordingly, thispromoter could be used to drive the expression of any desirable geneproducts to improve crop quality or enhance specific traits. As thetobacco nicotine demethylase promoter (e.g., SEQ ID NO:6) is inducibleand expressed during a particular period of the plant's life cycle,constructs containing this promoter can be introduced to the plant toexpress unique genes involved in the biosynthesis of flavor and aromaproducts that result from secondary metabolites. Examples of suchcompounds are compounds in the terpenoid pathway, other alkaloids, planthormones, flavonoids, or sugar-containing moieties. A tobacco nicotinedemethylase promoter may also be used to increase or modify theexpression of structural carbohydrates or proteins that affect end-useproperties. Further, a tobacco nicotine demethylase promoter could alsobe combined with heterologous genes that include genes involved in thebiosynthesis of nutritional products, pharmaceutical agents, orindustrial materials. The promoter may be used to drive thedown-regulation of genes endogenous to tobacco including nicotinedemethylase or other genes involved in alkaloid biosynthesis and or inother pathways.

Moreover, the promoter region (e.g., SEQ ID NO:6) of a tobacco nicotinedemethylase genes or the 3′ UTR (e.g., SEQ ID NO:7) of a tobacconicotine demethylase gene may also be used in any site-directed genesilencing methods such as T-DNA tagging, gene trapping and homologousrecombination to alter the expression pattern of a target gene, asdescribed herein. Promoter motifs, which can readily be identified in apromoter sequence, such as the sequence of SEQ ID NO:6 using standardmethods in the art, may also be used to identify factors that associatewith or regulate the expression of a tobacco nicotine demethylase.Desirably, a tobacco nicotine demethylase promoter region or othertranscriptional regulatory region is used to alter chemical propertiessuch as nornicotine content and nitrosamine levels in a plant.

Furthermore, any portion of a tobacco nicotine demethylase gene can beused as a genetic marker to isolate related genes, promoters orregulatory regions, or for screening for the demethylase gene in othertobacco or Nicotiana species.

Products

Tobacco products having a reduced amount of nitrosamine content aremanufactured using any of the tobacco plant material described hereinaccording to standard methods known in the art. In one embodiment,tobacco products are manufactured using plant material obtained from agenetically modified cured tobacco having a reduced amount ofnornicotine or NNN of less than about 5 mg/g, 4.5 mg/g, 4.0 mg/g, 3.5mg/g, 3.0 mg/g, 2.5 mg/g, 2.0 mg/g, 1.5 mg/g, 1.0 mg/g, 750 μg/g, 500μg/g, 250 μg/g, 100 μg/g, 75 μg/g, 50 μg/g, 25 μg/g, 10 μg/g, 7.0 μg/g,5.0 μg/g, 4.0 μg/g, 2.0 μg/g, 1.0 μg/g, 0.5 μg/g, 0.4 μg/g, 0.2 μg/g,0.1 μg/g, 0.05 μg/g, or 0.01 μg/g or wherein the percentage of secondaryalkaloids relative to total alkaloid content contained therein is lessthan 90%, 70%, 50%, 30%, 10%, 5%, 4%, 3%, 2%, 1.5%, 1%, 0.75%, 0.5%,0.25%, or 0.1%. That is, the cured tobacco is made from a geneticallymodified tobacco plant. The phrase “a reduced amount” is intended torefer to an amount of nornicotine or NNN or both in a transgenic tobaccoplant, tobacco or a tobacco product that is less than what would befound in a naturally-occurring tobacco plant, tobacco or a tobaccoproduct from the same variety of tobacco, processed in the same manner,which was not made transgenic material for reduced nornicotine or NNN.Thus, in some contexts, a naturally-occurring tobacco of the samevariety that has been processed in the same manner is used as a controlby which to measure whether a reduction of nornicotine or NNN has beenobtained by the methods described herein. Levels of nornicotine and NNNare measured according to methods well known in the tobacco art.

The following examples illustrate methods for carrying out the inventionand should be understood to be illustrative of, but not limiting upon,the scope of the invention which is defined in the appended claims.

Example 1 Development of Plant Tissue and Ethylene Treatment

Plant Growth

Plants were seeded in pots and grown in a greenhouse for 4 weeks. The4-week old seedlings were transplanted into individual pots and grown inthe greenhouse for 2 months. The plants were watered 2 times a day withwater containing 150 ppm NPK fertilizer during growth. The expandedgreen leaves were detached from plants to do the ethylene treatmentdescribed below.

Cell Line 78379

Tobacco line 78379, which is a burley tobacco line released by theUniversity of Kentucky was used as a source of plant material. Onehundred plants were cultured as standard in the an of growing tobacco,transplanted, and tagged with a distinctive number (1-100).Fertilization and field management were conducted as recommended.

Three quarters of the 100 plants converted between 20 and 100% of thenicotine to nornicotine. One quarter of the 100 plants converted lessthan 5% of the nicotine to nornicotine. Plant number 87 had the leastconversion (2%) while plant number 21 had 100% conversion. Plantsconverting less than 3% were classified as non-converters.Self-pollinated seed of plant number 87 and plant number 21, as well ascrossed (21×87 and 87×21) seeds were made to study genetic andphenotypic differences. Plants from selfed 21 were converters, and 99%of selfs from 87 were non-converters. The other 1% of the plants from 87showed low conversion (5-15%). Plants from reciprocal crosses were allconverters.

Cell Line 4407

Nicotiana line 4407, which is a burley line, was used as a source ofplant material. Uniform and representative plants (100) were selectedand tagged. Of the 100 plants 97 were non-converters and three wereconverters. Plant number 56 had the least amount of conversion (1.2%)and plant number 58 had the highest level of conversion (96%).Self-pollinated seeds and crossed seeds were made with these two plants.

Plants from selfed-58 segregated with 3:1 converter to non-converterratio. Plants 58-33 and 58-25 were identified as homozygous converterand nonconverter plant lines, respectively. The stable conversion of58-33 was confirmed by analysis of its progeny.

Cell Line PBLB01

PBLB01 is a burley line developed by ProfiGen, Inc. and was used as asource of plant material. The converter plant was selected fromfoundation seeds of PBLB01.

Ethylene Treatment Procedures

Green leaves were detached from 2-3 month greenhouse grown plants andsprayed with 0.3% ethylene solution (Prep brand Ethephon(Rhone-Poulenc)). Each sprayed leaf was hung in a curing rack equippedwith humidifier and covered with plastic. During the treatment, thesample leaves were periodically sprayed with the ethylene solution.Approximately 24-48 hour post ethylene treatment, leaves were collectedfor RNA extraction. Another sub-sample was taken for metabolicconstituent analysis to determine the concentration of leaf metabolitesand more specific constituents of interest such as a variety ofalkaloids.

As an example, alkaloids analysis could be performed as follows. Samples(0.1 g) were shaken at 150 rpm with 0.5 ml 2N NaOH, and a 5 mlextraction solution which contained quinoline as an internal standardand methyl t-butyl ether. Samples were analyzed on a HP 6890 GC equippedwith a FID detector. A temperature of 250° C. was used for the detectorand injector. An HP column (30 m-0.32 nm-1 mm) consisting of fusedsilica crosslinked with 5% phenol and 95% methyl silicon was used at atemperature gradient of 110-185° C. at 10° C. per minute. The column wasoperated at 100° C. with a flow rate of 1.7 cm³ min⁻¹ with a split ratioof 40:1 with a 2:1 injection volume using helium as the carrier gas.

Example 2 RNA Isolation

For RNA extractions, middle leaves from two-month old greenhouse grownplants were treated with ethylene as described above. The 0 and 24-48hours samples were used for RNA extraction. In some cases, leaf samplesunder the senescence process were taken from the plants 10 days postflower-head removal. These samples were also used for extraction. TotalRNA was isolated using Rneasy Plant Mini Kit® (Qiagen, Inc., Valencia,Calif.) according to the manufacturer's protocol.

The tissue sample was ground under liquid nitrogen to a fine powderusing a DEPC treated mortar and pestle. Approximately 100 milligrams ofground tissue were transferred to a sterile 1.5 ml Eppendorf tube. Thissample tube was placed in liquid nitrogen until all samples werecollected. Then, 45 μl of Buffer RLT as provided in the kit (with theaddition of Mercaptoethanol) was added to each individual tube. Thesample was vortexed vigorously and incubated at 56° C. for 3 minutes.The lysate was then applied to the QIAshredder® spin column sitting in a2 ml collection tube, and centrifuged for 2 minutes at maximum speed.The flow through was collected and 0.5 volume of ethanol was added tothe cleared lysate. The sample was mixed well and transferred to anRneasy® mini spin column sitting in a 2 ml collection tube. The samplewas centrifuged for 1 minute at 10,000 rpm. Next, 70 μl of buffer RW1was pipetted onto the Rneasy® column and centrifuged for 1 minute at10,000 rpm. Buffer RPE was pipetted onto the Rneasy® column in a newcollection tube and centrifuged for 1 minute at 10,000 rpm. Buffer RPEwas again, added to the Rneasy® spin column and centrifuged for 2minutes at maximum speed to dry the membrane. To eliminate any ethanolcarry over, the membrane was placed in a separate collection tube andcentrifuged for an additional 1 minute at maximum speed. The Rneasy®column was transferred into a new 1.5 ml collection tube, and 40 μl ofRnase-free water was pipetted directly onto the Rneasy® membrane. Thisfinal elute tube was centrifuged for 1 minute at 10,000 rpm. Quality andquantity of total RNA was analyzed by denatured formaldehyde gel andspectrophotometer.

Poly(A)RNA was isolated using Oligotex® poly A+ RNA purification kit(Qiagen Inc.) following the manufacture's protocol. About 200 μg totalRNA in 250 μl maximum volume was used. A volume of 250 μl of Buffer OBBand 15 μl of Oligotex® suspension was added to the 250 μl of total RNA.The contents were mixed thoroughly by pipetting and incubated for 3minutes at 70° C. on a heating block. The sample was then placed at roomtemperature for approximately 20 minutes. The Oligotex®: mRNA complexwas pelleted by centrifugation for 2 minutes at maximum speed. All but50 μl of the supernatant was removed from the microcentrifuge tube. Thesample was treated further by OBB buffer. The Oligotex®: mRNA pellet wasresuspended in 400 μl of Buffer OW2 by vortexing. This mix wastransferred onto a small spin column placed in a new tube andcentrifuged for 1 minute at maximum speed. The spin column wastransferred to a new tube and an additional 400 μl of Buffer OW2 wasadded to the column. The tube was then centrifuged for 1 minute atmaximum speed. The spin column was transferred to a final 1.5 mlmicrocentrifuge tube. The sample was eluted with 60 μl of hot (70° C.)Buffer OEB. Poly A product was analyzed by denatured formaldehyde gelsand spectrophotometric analysis.

Example 3 Reverse-Transcription-PCR

First strand cDNA was produced using SuperScript reverse transcriptasefollowing the manufacturer's protocol (Invitrogen, Carlsbad, Calif.).The poly A+ enriched RNA/oligo dT primer mix consisted of less than 5 μgof total RNA, 1 μl of 10 mM dNTP mix, 1 μl of Oligo d(T)₁₂ _(—) ₁₈ (0.5μg/μl), and up to 10 μl of DEPC-treated water. Each sample was incubatedat 65° C. for 5 minutes, then placed on ice for at least 1 minute. Areaction mixture was prepared by adding each of the following componentsin order: 2 μl 10×RT buffer, 4 μl of 25 mM MgCl₂, 2 μl of 0.1 M DTT, and1 μl of RNase OUT Recombinant RNase Inhibitor. An addition of 9 μl ofreaction mixture was pipetted to each RNA/primer mixture and gentlymixed. It was incubated at 42° C. for 2 minutes and 1 μl of Super ScriptII RT was added to each tube. The tube was incubated for 50 minutes at42° C. The reaction was terminated at 70° C. for 15 minutes and chilledon ice. The sample was collected by centrifugation and 1 μl of RNase Hwas added to each tube and incubated for 20 minutes at 37° C. The secondPCR was carried out with 200 pmoles of forward primer and 100 pmolesreverse primer (mix of 18 nt oligo d(T) followed by 1 random base).

Reaction conditions were 94° C. for 2 minutes and then 40 cycles of PCRat 94° C. for 1 minute, 45° C. to 60° C. for 2 minutes, 72° C. for 3minutes, with a 72° C. extension for an extra 10 min. Ten microliters ofthe amplified sample were analyzed by electrophoresis using a 1% agarosegel. The correct size fragments were purified from agarose gel.

Example 4 Generation of PCR Fragment Populations

PCR fragments from Example 3 were ligated into a pGEM-T Easy Vector(Promega, Madison, Wis.) following the manufacturer's instructions. Theligated product was transformed into JM 109 competent cells and platedon LB media plates for blue/white selection. Colonies were selected andgrown in a 96 well plate with 1.2 ml of LB media overnight at 37° C.Frozen stock was generated for all selected colonies. Plasmid DNA waspurified from plates using Beckman's Biomeck 2000 miniprep robotics withWizard SV Miniprep kit (Promega). Plasmid DNA was eluted with 100 μlwater and stored in a 96 well plate. Plasmids were digested by EcoR1 andwere analyzed using 1% agarose gel to confirm the DNA quantity and sizeof inserts. Plasmids containing a 400-600 bp insert were sequenced usinga CEQ 2000 sequencer (Beckman, Fullerton, Calif.). The sequences werealigned with GenBank database by BLAST search. The p450 relatedfragments were identified and further analyzed. Alternatively, p450fragments were isolated from subtraction libraries. These fragments werealso analyzed as described above.

Example 5 cDNA Library Construction

A cDNA library was constructed by preparing total RNA from ethylenetreated leaves as follows. First, total RNA was extracted from ethylenetreated leaves of tobacco line 58-33 using a modified acid phenol andchloroform extraction protocol. The protocol was modified to use onegram of tissue that was ground and subsequently vortexed in 5 ml ofextraction buffer (100 mM Tris-HCl, pH 8.5; 200 mM NaCl; 10 mM EDTA;0.5% SDS) to which 5 ml phenol (pH5.5) and 5 ml chloroform was added.The extracted sample was centrifuged and the supernatant was saved. Thisextraction step was repeated 2-3 times until the supernatant appearedclear. Approximately 5 ml of chloroform was added to remove traceamounts of phenol. RNA was precipitated from the combined supernatantfractions by adding a 3-fold volume of ethanol and 1/10 volume of 3MNaOAc (pH5.2) and storing at −20° C. for 1 hour. After transfer to aCorex glass container the RNA fraction was centrifuged at 9,000 RPM for45 minutes at 4° C. The pellet was washed with 70% ethanol and spun for5 minutes at 9,000 RPM at 4° C. After drying the pellet, the pelletedRNA was dissolved in 0.5 ml RNase free water. The quality and quantityof total RNA was analyzed by denatured formaldehyde gel andspectrophotometer, respectively.

The resultant total RNA was used to isolate poly A+ RNA using anOligo(dT) cellulose protocol (Invitrogen) and microcentrifuge spincolumns (Invitrogen) by the following protocol. Approximately twenty mgof total RNA was twice subjected to purification to obtain high qualitypoly A+ RNA. Poly A+ RNA product was analyzed by performing denaturedformaldehyde gel and subsequent RT-PCR of known full-length genes toensure high quality of mRNA.

Next, poly A+ RNA was used as template to produce a cDNA libraryemploying cDNA synthesis kit, ZAP-cDNA synthesis kit, and ZAP-cDNAGigapack III gold cloning kit (Stratagene, La Jolla, Calif.). The methodinvolved following the manufacture's protocol as specified.Approximately 8 μg of poly A+ RNA was used to construct cDNA library.Analysis of the primary library revealed about 2.5×10⁶-1×10⁷ pfu. Aquality background test of the library was completed by complementationassays using IPTG and X-gal, where recombinant plaques was expressed atmore than 100-fold above the background reaction.

A more quantitative analysis of the library by random PCR showed thataverage size of insert cDNA was approximately 1.2 kb. The method used atwo-step PCR method. For the first step, reverse primers were designedbased on the preliminary sequence information obtained from p450fragments. The designed reverse primers and T3 (forward) primers wereused to amplify corresponding genes from the cDNA library. PCR reactionswere subjected to agarose electrophoresis and the corresponding bands ofhigh molecular weight were excised, purified, cloned and sequenced. Inthe second step, new primers designed from 5′UTR or the start codingregion of p450 as the forward primers together with the reverse primers(designed from 3′UTR of p450) were used in the subsequent PCR to obtainfull-length p450 clones.

The p450 fragments were generated by PCR amplification from theconstructed cDNA library as described in Example 3 with the exception ofthe reverse primer. The T7 primer located on the plasmid downstream ofcDNA inserts was used as a reverse primer. PCR fragments were isolated,cloned and sequenced as described in Example 4.

Full-length p450 genes were isolated by this PCR method from constructedcDNA library. Gene specific reverse primers (designed from thedownstream sequence of p450 fragments) and a forward primer (T3 onlibrary plasmid) were used to clone the full-length genes. PCR fragmentswere isolated, cloned and sequenced. If necessary, a second PCR step wasapplied. In the second step, new forward primers designed from 5′UTR ofcloned p450s together with the reverse primers designed from 3′UTR ofp450 clones were used in the subsequent PCR reactions to obtainfull-length p450 clones. The clones were subsequently sequenced.

Example 6 Characterization of Cloned Fragments Reverse Southern BlottingAnalysis

Nonradioactive large-scale reverse Southern blotting assays wereperformed on all p450 clones identified in above examples to detect thedifferential expression. It was observed that the level of expressionamong different p450 clusters was very different. Further real timedetection was conducted on those with high expression.

Nonradioactive Southern blotting procedures were conducted as follows.

1) Total RNA was extracted from ethylene treated and nontreatedconverter (58-33) and nonconverter (58-25) leaves using the QiagenRnaeasy kit as described in Example 2.

2) A probe was produced by biotin-tail labeling a single strand cDNAderived from poly A+ enriched RNA generated in above step. This labeledsingle strand cDNA was generated by RT-PCR of the converter andnonconverter total RNA (Invitrogen) as described in Example 3 with theexception of using biotinylated oligo dT as a primer (Promega). Thesewere used as a probe to hybridize with cloned DNA.

3) Plasmid DNA was digested with restriction enzyme EcoR1 and run onagarose gels. Gels were simultaneously dried and transferred to twonylon membranes (Biodyne B). One membrane was hybridized with converterprobe and the other with nonconverter probe. Membranes wereUV-crosslinked (auto crosslink setting, 254 nm, Stratagene,Stratalinker) before hybridization.

Alternatively, the inserts were PCR amplified from each plasmid usingthe sequences located on both arms of p-GEM plasmid, T3 and SP6, asprimers. The PCR products were analyzed by running on a 96 wellReady-to-run agarose gels. The confirmed inserts were dotted on twonylon membranes. One membrane was hybridized with converter probe andthe other with nonconverter probe.

4) The membranes were hybridized and washed following the manufacture'sinstructions with the modification of washing stringency (Enzo MaxSencekit, Enzo Diagnostics, Inc, Farmingdale, N.Y.). The membranes wereprehybridized with hybridization buffer (2×SSC buffered formamide,containing detergent and hybridization enhancers) at 42° C. for 30 minand hybridized with 10 μl denatured probe overnight at 42° C. Themembranes then were washed in 1× hybridization wash buffer 1 time atroom temperature for 10 min and 4 times at 68° C. for 15 min. Themembranes were ready for the detection procedure.

5) The washed membranes were detected by alkaline phosphatase labelingfollowed by NBT/BCIP colometric detection as described in manufacture'sdetection procedure (Enzo Diagnostics, Inc.). The membranes were blockedfor one hour at room temperature with 1× blocking solution, washed 3times with 1× detection reagents for 10 min, washed 2 times with 1×predevelopment reaction buffer for 5 min and then developed the blots indeveloping solution for 30-45 min until the dots appear. All reagentswere provided by the manufacturer (Enzo Diagnostics, Inc). In addition,large-scale reverse Southern assay was also performed using KPL Southernhybridization and detection kit following the manufacturer'sinstructions (KPL, Gaithersburg, Md.).

Example 7 Characterization of Clones Northern Blot Analysis

As an alternative to Southern blot analysis, some membranes werehybridized and detected as described in the example of Northern blottingassays. Northern hybridization was used to detect mRNA differentiallyexpressed in Nicotiana as follows.

A random priming method was used to prepare probes from cloned p450(Megaprime DNA Labelling Systems, Amersham Biosciences). The followingcomponents were mixed: 25 ng denatured DNA template; 4 ul of eachunlabeled dTTP, dGTP and dCTP; 5 ul of reaction buffer; P³²-labelleddATP and 2 ul of Klenow I; and H20, to bring the reaction to 5 μl. Themixture was incubated in 37° C. for 1-4 hours, and stopped with 2 μl of0.5 M EDTA. The probe was denatured by incubation at 95° C. for 5minutes before use.

RNA samples were prepared from ethylene treated and non-treated freshleaves of several pairs of tobacco lines. In some cases poly A+ enrichedRNA was used. Approximately 15 μg total RNA or 1.8 μg mRNA (methods ofRNA and mRNA extraction as described in Example 5) were brought to equalvolume with DEPC H2O (5-10 μl). The same volume of loading buffer(1×MOPS; 18.5% Formaldehyde; 50% Formamide; 4% Fico11400;Bromophenolblue) and 0.5 μl EtBr (0.5 μg/μl) were added. The sampleswere subsequently denatured in preparation for separation of the RNA byelectrophoresis.

Samples were subjected to electrophoresis on a formaldehyde gel (1%Agarose, 1×MOPS, 0.6 M Formaldehyde) with 1×MOP buffer (0.4 MMorpholinopropanesulfonic acid; 0.1 M Na-acetate-3×H2O; 10 mM EDTA;adjust to pH 7.2 with NaOH). RNA was transferred to a Hybond-N+ membrane(Nylon, Amersham Pharmacia Biotech) by capillary method in 10×SSC buffer(1.5 M NaCl; 0.15 M Na-citrate) for 24 hours. Membranes with RNA sampleswere UV-crosslinked (auto crosslink setting, 254 nm, Stratagene,Stratalinker) before hybridization.

The membrane was prehybridized for 1-4 hours at 42° C. with 5-10 mlprehybridization buffer (5×SSC; 50% Formamide; 5×Denhardt's-solution; 1%SDS; 1001·tg/ml heat-denatured sheared non-homologous DNA). Oldprehybridization buffer was discarded, and new prehybridization bufferand probe were added. The hybridization was carried out overnight at 42°C. The membrane was washed for 15 minutes with 2×SSC at roomtemperature, followed by a wash with 2×SSC.

Northern analysis was performed using full-length clones on tobaccotissue obtained from converter and nonconverter burley lines that wereinduced by ethylene treatment. The purpose was to identify thosefull-length clones that showed elevated expression in ethylene inducedconverter lines relative to ethylene induced converter lines relative toethylene induced nonconverter burley lines. By so doing, thefunctionality relationship of full-length clones may be determined bycomparing biochemical differences in leaf constituents between converterand nonconverter lines.

Example 8 Immunodetection of p450s Encoded by the Cloned Genes

Peptide regions corresponding to 20-22 amino acids in length from threep450 clones were selected for 1) having lower or no homology to otherclones and 2) having good hydrophilicity and antigenicity. The aminoacid sequences of the peptide regions selected from the respective p450clones are listed below. The synthesized peptides were conjugated withKHL and then injected into rabbits. Antisera were collected 2 and 4weeks after the 4′ h injection (Alpha Diagnostic Intl. Inc. San Antonio,Tex.).

D234-AD1 DIDGSKSKLVKAHRKIDEILG (SEQ ID NO: 8) D90a-BB3RDAFREKETFDENDVEELNY (SEQ ID NO: 9) D89-AB 1 FKNNGDEDRHFSQKLGDLADKY(SEQ ID NO: 10)

Antisera were examined for crossreactivity to target proteins fromtobacco plant tissue by Western Blot analysis. Crude protein extractswere obtained from ethylene treated (0 to 40 hours) middle leaves ofconverter and nonconverter lines. Protein concentrations of the extractswere determined using RC DC Protein Assay Kit (BIO-RAD) following themanufacturer's protocol.

Two micrograms of protein were loaded onto each lane and the proteinswere separated on 10%-20% gradient gels using the Laemmli SDS-PAGEsystem. The proteins were transferred from gels to PROTRANNitrocellulose Transfer Membranes (Schleicher & Schuell) with theTrans-Blot Semi-Dry cell (BIO-RAD). Target p450 proteins were detectedand visualized with the ECL Advance Western Blotting Detection Kit(Amersham Biosciences). Primary antibodies against the synthetic-KLHconjugates were made in rabbits. Secondary antibody against rabbit IgG,coupled with peroxidase, was purchased from Sigma. Both primary andsecondary antibodies were used at 1:1000 dilutions. Antibodies showedstrong reactivity to a single band on the Western Blots indicating thatthe antisera were monospecific to the target peptide of interest.Antisera were also crossreactive with synthetic peptides conjugated toKLH.

Example 9 Nucleic Acid Identity and Structure Relatedness of IsolatedNucleic Acid Fragments

Over 100 cloned p450 fragments were sequenced in conjunction withNorthern blot analysis to determine their structural relatedness. Theapproach used forward primers based either of two common p450 motifslocated near the carboxyl-terminus of the p450 genes. The forwardprimers corresponded to cytochrome p450 motifs FXPERF (SEQ ID NO:11) orGRRXCP(A/G) (SEQ ID NO:12). The reverse primers used standard primersfrom either the plasmid, SP6 or T7 located on both arms of pGEM plasmid,or a poly A tail. The protocol used is described below.

Spectrophotometry was used to estimate the concentration of startingdouble stranded DNA following the manufacturer's protocol (BeckmanCoulter). The template was diluted with water to the appropriateconcentration, denatured by heating at 95° C. for 2 minutes, andsubsequently placed on ice. The sequencing reaction was prepared on iceusing 0.5 to 10 μl of denatured DNA template, 2 μl of 1.6 pmole of theforward primer, 8 μl of DTCS Quick Start Master Mix and the total volumebrought to 20 μl with water. The thermocycling program consisted of 30cycles of the follow cycle: 96° C. for 20 seconds, 50° C. for 20seconds, and 60° C. for 4 minutes followed by holding at 4° C.

The sequencing reaction was stopped by adding 5 μl of stop buffer (equalvolume of 3M NaOAc and 100 mM EDTA and 1 μl of 20 mg/ml glycogen). Thesample was precipitated with 60 μl of cold 95% ethanol and centrifugedat 6000×g for 6 minutes. Ethanol was discarded. The pellet was 2 washeswith 200 μl of cold 70% ethanol. After the pellet was dry, 40 μl of SLSsolution was added and the pellet was resuspended. A layer of mineraloil was over laid. The sample was then, placed on the CEQ 8000 AutomatedSequencer for further analysis.

In order to verify nucleic acid sequences, nucleic acid sequence wasre-sequenced in both directions using forward primers to the FXPERF (SEQID NO:11) or GRRXCP(A/G) (SEQ ID NO:12) region of the p450 gene orreverse primers to either the plasmid or poly A tail. All sequencing wasperformed at least twice in both directions.

The nucleic acid sequences of cytochrome p450 fragments were compared toeach other from the coding region corresponding to the first nucleicacid after the region encoding the GRRXCP(AIG) (SEQ ID NO:12) motifthrough to the stop codon. This region was selected as an indicator ofgenetic diversity among p450 proteins. A large number of geneticallydistinct p450 genes, in excess of 70 genes, were observed, similar tothat of other plant species. Upon comparison of nucleic acid sequences,it was found that the genes could be placed into distinct sequencesgroups based on their sequence identity. It was found that the bestunique grouping of p450 members was determined to be those sequenceswith 75% nucleic acid identity or greater. (See e.g., Table 1 of the US2004/0162420 patent application publication, which is incorporatedherein by reference.) Reducing the percentage identity resulted insignificantly larger groups. A preferred grouping was observed for thosesequences with 81% nucleic acid identity or greater, a more preferredgrouping 91% nucleic acid identity or greater, and a most preferredgrouping for those sequences 99% nucleic acid identity of greater. Mostof the groups contained at least two members and frequently three ormore members. Others were not repeatedly discovered suggesting thatapproach taken was able to isolated both low and high expressing mRNA inthe tissue used.

Using GeneChip technology to identify genes that are differentiallyexpressed in converter versus non-converter tobacco lines, it wasdetermined that D121-AA8 had reproducible induction in ethylene-treatedconverter lines. Based on these results, the D121-AA8 gene (the cDNAsequence of which is the sequence of SEQ ID NO:3; FIG. 3) was identifiedas the tobacco nicotine demethylase gene of interest.

In view of the p450 nomenclature rule, the tobacco nicotine demethylasegene is novel and belongs to CYP82E class (The Arabidopsis GenomeInitiative (AGI) and The Arabidopsis Information Resource (TAIR); Frank,Plant Physiol. 110:1035-1046, 1996; Whitbred et al., Plant Physiol.124:47-58, 2000); Schopfer and Ebel, Mol. Gen. Genet. 258:315-322, 1998;and Takemoto et al., Plant Cell Physiol. 40:1232-1242, 1999).

Example 10 Biochemical Analysis of the Tobacco Nicotine Demethylase

Biochemical analysis, for example, as described in previously filedapplications that are incorporated herein by reference, determined thatthe sequence of SEQ ID NO:3 encodes a tobacco nicotine demethylase (SEQID NO:4; FIG. 3).

In particular, the function of the candidate clone (D121-AA8), wasconfirmed as the coding gene for nicotine demethylase, by assayingenzyme activity of heterologously expressed p450 in yeast cells asfollows.

1. Construction of Yeast Expression Vector

The putative protein-coding sequence of the tobacco nicotinedemethylase-encoding cDNA (D 121 AA8), was cloned into the yeastexpression vector pYeDP60. Appropriate BamHI and Mfel sites (underlinedbelow) were introduced via PCR primers containing these sequences eitherupstream of the translation start codon (ATG) or downstream of the stopcodon (TAA). The Mfel on the amplified PCR product is compatible withthe EcoRI site on the vector. The primers used to amplify the cDNA were5′-TAGCTACGCGGATCCATGCTTTCTCCCATAGAAGCC-3′ (SEQ ID NO:27) and5′-CTGGATCACAATTGTTAGTGATGGTGATGGTGATGCGATCCTCTATAAAGCTCAGGT GCCAGGC-3′(SEQ ID NO:28). A segment of sequence coding nine extra amino acids atthe C-terminus of the protein, including six histidines, wasincorporated into the reverse primer to facilitate expression of 6-Histagged p450 upon induction. PCR products were ligated into pYeDP60vector after enzyme digestions in the sense orientation with referenceto the GAL10-CYC 1 promoter. Proper construction of the yeast expressionvectors was verified by restriction enzyme analysis and DNA sequencing.

2. Yeast Transformation

The WAT11 yeast line, modified to express Arabidopsis NADPH-cytochromep450 reductase ATR1, was transformed with the pYeDP60-p450 cDNAplasmids. Fifty micro-liters of WAT11 yeast cell suspension was mixedwith −1 μg plasmid DNA in a cuvette with 0.2 cm electrode gap. One pulseat 2.0 kV was applied by an Eppendorf electroporator (Model 2510). Cellswere spread onto SGI plates (5 g/L bactocasamino acids, 6.7 g/L yeastnitrogen base without amino acids, 20 g/L glucose, 40 mg/LDL-tryptophan, 20 g/L agar). Transformants were confirmed by PCRanalysis performed directly on randomly selected colonies.

3. p450 Expression in Transformed Yeast Cells

Single yeast colonies were used to inoculate 30 mL SGI media (5 g/Lbactocasamino acids, 6.7 g/L yeast nitrogen base without amino acids, 20g/L glucose, 40 mg/L DL-tryptophan) and grown at 30° C. for about 24hours. An aliquot of this culture was diluted 1:50 into 1000 mL of YPGEmedia (10 g/L yeast extract, 20 g/L bacto peptone, 5 g/L glucose, 30ml/L ethanol) and grown until glucose was completely consumed asindicated by the colorimetric change of a Diastix urinalysis reagentstrip (Bayer, Elkhart, Ind.). Induction of cloned P450 was initiated byadding DL-galactose to a final concentration of 2%. The cultures weregrown for an additional 20 hours before used for in vivo activity assayor for microsome preparation.

WATT 1 yeast cells expressing pYeDP60-CYP71D20 (a p450 catalyzing thehydroxylation of 5-epi-aristolochene and 1-deoxycapsidiol in Nicotianatabacum) were used as control for the p450 expression and enzymeactivity assays.

To evaluate the effectiveness of the yeast expression of the p450 ingreat detail, reduced CO difference spectroscopy was performed. Thereduced CO spectrum exhibited a peak at 450 nm proteins from all fourp450 transformed yeast lines. No similar peaks were observed in themicrosomes of the control yeast or the vector control yeast. The resultsindicated that p450 proteins were expressed effectively in yeast linesharboring the pYeDP60-CYP 450. Concentrations of expressed p450 proteinin yeast microsome ranged from 45 to 68 nmole/mg of total protein.

4. In Vivo Enzyme Assay

The nicotine demethylase activity in the transformed yeast cells wereassayed by feeding of yeast culture with DL-Nicotine(Pyrrolidine-2-¹⁴C). 14C labeled nicotine (54 mCilmmol) was added to 75μl of the galactose-induced culture for a final concentration of 55 μM.The assay culture was incubated with shaking in 14 ml polypropylenetubes for 6 hours and was extracted with 900 μl methanol. Afterspinning, 20 μl of the methanol extract was separated with an rp-HPLCand the nornicotine fraction was quantitated by LSC.

The control culture of WAT11 (pYeDP60-CYP71D20) did not convert nicotineto nornicotine, showing that the WAT11 yeast strain does not containendogenous enzyme activities that can catalyze the step of nicotinebioconversion to nornicotine. In contrast, yeast expressing the tobacconicotine demethylase gene produced detectable amount of nornicotine,indicating the nicotine demethylase activity of the translation productof SEQ ID NO:3.

5. Yeast Microsome Preparation

After induction by galactose for 20 hours, yeast cells were collected bycentrifugation and washed twice with TES-M buffer (50 mM Tris-HCl, pH7.5, 1 mM EDTA, 0.6 M sorbitol, 10 mM 2-mercaptoethanol). The pellet wasresuspended in extraction buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.6M sorbitol, 2 mM 2-mercaptoethanol, 1% bovine serum album, ProteaseInhibitor Cocktail (Roche) at 1 tablet/50 ml). Cells were then brokenwith glass beads (0.5 mm in diameter, Sigma) and the cell extract wascentrifuged for 20 min at 20,000×g to remove cellular debris. Thesupernatant was subjected to ultracentrifugation at 100,000×g for 60 minand the resultant pellet contained the microsomal fraction. Themicrosomal fraction was suspended in TEG-M buffer (50 mM Tris-HCl, pH7.5, 1 mM EDTA, 20% glycerol and 1.5 mM 2-mercaptoethanol) at proteinconcentration of 1 mg/mL. Microsomal preparations were stored in aliquid nitrogen freezer until use.

6. Enzyme Activity Assay in Yeast Microsomal Preparations

Nicotine demethylase activity assays with yeast microsomal preparationswere performed. In particular, DL-Nicotine (Pyrrolidine-2-¹⁴C) wasobtained from Moravek Biochemicals and had a specific activity of 54mCi/mmol. Chlorpromazine (CPZ) and oxidized cytochrome c (cyt. C), bothP450 inhibitors, were purchased from Sigma. The reduced form ofnicotinamide adenine dinucleotide phosphate (NADPH) is the typicalelectron donor for cytochrome P450 via the NADPH:cytochrome P450reductase. NADPH was omitted for control incubation. The routine enzymeassay included microsomal proteins (around 1 mg/ml), 6 mM NADPH, and 55μM ¹⁴C labeled nicotine. The concentration of CPZ and Cyt. C, when used,was 1 mM and 100 μM, respectively. The reaction was carried at 25° C.for 1 hour and was stopped with the addition of 300 μl methanol to each25 μl reaction mixture. After centrifugation, 20 μl of the methanolextract was separated with a reverse-phase High Performance LiquidChromatography (HPLC) system (Agilent) using an Inertsil ODS-3 3μ(150×4.6 mm) chromatography column from Varian. The isocratic mobilephase was the mixture of methanol and 50 mM potassium phosphate buffer,pH 6.25, with ratio of 60:40 (v/v) and the flow rate was 1 ml/min. Thenornicotine peak, as determined by comparison with authentic non-labelednornicotine, was collected and subjected to 2900 tri-carb LiquidScintillation Counter (LSC) (Perkin Elmer) for quantification. Theactivity of nicotine demethylase is calculated based on the productionof ¹⁴C labeled nornicotine over 1 hour incubation.

Microsomal preparations from control yeast cells expressing CYP71D20 didnot have any detectable microsomal nicotine demethylase activity. Incontrast, microsomal samples obtained from yeast cells expressing thetobacco nicotine demethylase gene showed significant levels of nicotinedemethylase activity. The nicotine demethylase activity required NADPHand was shown to be inhibited by p450 specific inhibitors, consistentwith tobacco nicotine demethylase being a p450. The enzyme activity fortobacco nicotine demethylase (D121-AA8) was approximately 10.8 pKat/mgprotein as calculated by radioactive intensity and proteinconcentrations. A typical set of enzyme assay results obtained for theyeast cells is shown in the Table 1.

TABLE 1 DEMETHYLASE ACTIVITY IN MICROSOMES OF YEAST CELLS EXPRESSINGD121-AA8 AND CONTROL P450 Microsomes + Microsomes + 1 mM chlor- 100 μMMicrosomes − Sample Microsomes promazine cytochrome C NADPH D121- 10.8 ±1.2* 1.4 ± 1.3 2.4 ± 0.7 0.4 ± 0.1 AA8 pkat/mg pkat/mg pkat/mg pkat/mgprotein protein protein protein Control Not Detected Not Detected NotDetected Not Detected *Average results of 3 replicates.

Together these experiments demonstrated that the cloned full-lengthtobacco nicotine demethylase (SEQ ID NO:3; D121-AA8) encodes acytochrome p450 protein that catalyzes the conversion of nicotine tonornicotine when expressed in yeast.

Example 11 Related Amino Acid Sequence Identity of Isolated Nucleic AcidFragments

The amino acid sequences of nucleic acid sequences obtained forcytochrome p450 fragments from Example 8 were deduced. The deducedregion corresponded to the amino acid immediately after the GXRXCP(A/G)(SEQ ID NO:13) sequence motif to the end of the carboxyl-terminus, orstop codon. Upon comparison of sequence identity of the fragments, aunique grouping was observed for those sequences with 70% amino acididentity or greater. A preferred grouping was observed for thosesequences with 80% amino acid identity or greater, more preferred with90% amino acid identity or greater, and a most preferred grouping forthose sequences 99% amino acid identity of greater. Several of theunique nucleic acid sequences were found to have complete amino acididentity to other fragments and therefore only one member with theidentical amino acid was reported.

At least one member of each amino acid identity group was selected forgene cloning and functional studies using plants. In addition, groupmembers that are differentially affected by ethylene treatment or otherbiological differences as assessed by Northern and Southern analysiswere selected for gene cloning and functional studies. To assist in genecloning, expression studies and whole plant evaluations, peptidespecific antibodies can be prepared based on sequence identity anddifferential sequence.

Example 12 Related Amino Acid Sequence Identity of Full-Length Clones

The nucleic acid sequence of full-length Nicotiana genes cloned inExample 5 were deduced for their entire amino acid sequence. Cytochromep450 genes were identified by the presence of three conserved p450domain motifs, which corresponded to UXXRXXZ (SEQ ID NO:14), PXRFXF (SEQID NO:15) or GXRXC (SEQ ID NO:16) at the carboxyl-terminus where U is Eor K, X is any amino acid and Z is P, T, S or M. All p450 genes werecharacterized for amino acid identity using a BLAST program comparingtheir full-length sequences to each other and to known tobacco genes.The program used the NCBI special BLAST tool (Align two sequences(b12seq), http://www.ncbi.nlm.nih.gov/blast/b12seq/b12.html). Twosequences were aligned under BLASTN without filter for nucleic acidsequences and BLASTP for amino acid sequences. Based on their percentageamino acid identity, each sequence was grouped into identity groupswhere the grouping contained members that shared at least 85% identitywith another member. A preferred grouping was observed for thosesequences with 90% amino acid identity or greater, a more preferredgrouping had 95% amino acid identity or greater, and a most preferredgrouping had those sequences 99% amino acid identity or greater. Theamino acid sequence of the full-length nicotine demethylase gene wasdeduced to have the sequence provided in SEQ ID NO:4 (FIG. 3).

Example 13 Nicotiana Cytochrome P450 Clones Lacking One or More of theTobacco P450 Specific Domains

Four clones had high nucleic acid homology, ranging 90% to 99% nucleicacid homology. However, due to a nucleotide frameshift these genes didnot contain one or more of three C-terminus cytochrome p450 domains andwere excluded from identity groups.

Example 14 Use of Nicotiana Cytochrome P450 Fragments and Clones inAltered Regulation of Tobacco Qualities

The use of tobacco p450 nucleic acid fragments or whole genes are usefulin identifying and selecting those plants that have altered tobaccophenotypes or tobacco constituents and, more importantly, alteredmetabolites. Transgenic tobacco plants are generated by a variety oftransformation systems that incorporate nucleic acid fragments orfull-length genes, selected from those reported herein, in orientationsfor either down-regulation, for example anti-sense orientation, orover-expression for example, sense orientation and the like. Forover-expression to full-length genes, any nucleic acid sequence thatencodes the entire or a functional part or amino acid sequence of thefull-length genes described in this invention is desirable. Such nucleicacid sequences desirably are effective for increasing the expression ofa certain enzyme and thus resulting in phenotypic effect withinNicotiana. Nicotiana lines that are homozygous are obtained through aseries of backcrossing and assessed for phenotypic changes including,but not limited to, analysis of endogenous p450 RNA, transcripts, p450expressed peptides and concentrations of plant metabolites usingtechniques commonly available to one having ordinary skill in the art.The changes exhibited in the tobacco plants provide information on thefunctional role of the selected gene of interest or are of use aspreferred Nicotiana plant species.

Example 15 Cloning of the Genomic Tobacco Nicotine Demethylase fromConverter Burley Tobacco

Genomic DNA was extracted from converter Burley tobacco plant line4407-33 (a Nicotiana tabacum variety 4407 line) using Qiagen Plant Easykit as described in above Examples (see also the manufacturer'sprocedure).

The primers were designed based on the 5′ promoter and 3′ UTR regioncloned in previous examples. The forward primers were 5′-GGC TCT AGA TAAATC TCT TAA GTT ACT AGG TTC TAA-3′ (SEQ ID NO:17) and 5′-TCT CTA AAG TCCCCT TCC-3′ (SEQ ID NO:25) and the reverse primers were 5′-GGC TCT AGAAGT CAA TTA TCT TCT ACA AAC CTT TAT ATA TTA GC-3′ (SEQ ID NO:18), and5′-CCA GCA TTC CTC AAT TTC-3′ (SEQ ID NO:26). PCR was applied to the4407-33 genomic DNA with 100 μl of reaction mix. Pfx high fidelityenzyme was used for PCR amplification. The PCR product was visualized on1% agarose gel after electrophoresis. A single band with molecularweight of approximately 3.5 kb was observed and excised from the gel.The resulting band was purified using a gel purification kit (Qiagen;based on manufacturer's procedure). The purified DNA was digested byenzyme Xba I (NEB; used according to the manufacturer's instructions).The pBluescript plasmid was digested by Xba I using same procedure. Thefragment was gel purified and ligated to pBluescript plasmid. Theligation mix was transformed into competent cell GMIO9 and plated ontoLB plate containing 100 mg/l of ampicillin with blue/white selection.The white colonies were picked and grown into 10 ml LB liquid mediacontaining ampicillin. The DNA was extracted by miniprep. The plasmidDNA containing the insert was sequenced using a CEQ 2000 sequencer(Beckman, Fullerton, Calif.) based on the manufacturer's procedure. TheT3 and T7 primers and 8 other internal primers were used for sequencing.The sequence was assembled and analyzed, thus providing the genomicsequence (SEQ ID NO:1; FIGS. 2-1 to 2-3).

Comparison of the sequence of SEQ ID NO:1 with the sequence of SEQ IDNO:3 allowed the determination of a single intron within the codingportion of the gene (identified as the sequence of SEQ ID NO:5; FIG. 4).As shown in FIG. 1, the genomic structure of the tobacco nicotinedemethylase includes two exons flanking a single intron. The first exonspans nucleotides 2010 to 2949 of SEQ ID NO:1, which encode amino acids1-313 of SEQ ID NO:2, and the second exon spans nucleotides 3947 to 4562of SEQ ID NO:1, which encode amino acids 314-517 of SEQ ID NO:2.Accordingly, the intron spans nucleotides 2950-3946 of SEQ ID NO:1. Theintron sequence is provided in FIG. 4 and is that of SEQ ID NO:5. Thetranslation product of the genomic DNA sequence is provided in FIG. 2-1as the sequence of SEQ ID NO:2. The tobacco nicotine demethylase aminoacid sequence contains an endoplasmic reticulum membrane anchoringmotif.

Example 16 Cloning 5′ Flanking Sequences (SEQ ID NO:6) and 3′UTR (SEQ IDNO:7) from Converter Tobacco

A. Isolation of Total DNA from Converter Tobacco Leaves Tissue

Genomic DNA was isolated from leaves of converter tobacco 4407-33. Theisolation of DNA was performed using a DNeasy Plant Mini Kit from thecompany Qiagen, Inc. (Valencia, Calif.) according to the manufacturer'sprotocol. The manufacturer's manual Dneasy' Plant Mini and DNeasy PlantMaxi Handbook, Qiagen January 2004 is incorporated hereby as reference.The procedure for DNA preparation included the following steps: Tobaccoleaf tissue (approximately 20 mg dry weight) was ground to a fine powderunder liquid nitrogen for 1 minute. The tissue powder was transferredinto a 1.5 ml tube. Buffer AP1 (400 μl) and 4 μl of RNase stock solution(100 mg/ml) were added to a maximum of 100 mg of ground leaf tissue andvortexed vigorously. The mixture was incubated for 10 min at 65° C. andmixed 2-3 times during incubation by inverting tube. Buffer AP2 (130 μl)was then added to the lysate. The mixture was mixed and incubated for 5min on ice. The lysate was applied to a QlAshredder Mini Spin Column andcentrifuged for 2 min (14,000 rpm). The flow-through fraction wastransferred to a new tube without disturbing the cell-debris pellet.Buffer AP3/E (1.5 volumes) was then added to the cleared lysate andmixed by pipetting. The mixture (650 μl) from the preceding stepincluding any precipitate was applied to a DNeasy Mini Spin Column. Themixture was centrifuged for 1 min at >6000×g (>8000 rpm) and theflow-through was discarded. This was repeated with the remaining sampleand the flow-through and collection tube were discarded. DNeasy MiniSpin Column was placed in a new 2 ml collection tube. Then buffer AW(500 μl) was added to the DNeasy column and centrifuged for 1 min (>8000rpm). The flow-through was discarded. The collection tube was reused inthe next step. Buffer AW (500 μl) was then added to the DNeasy columnand centrifuged for 2 min (>14,000 rpm) in order to dry the membrane.The DNeasy column was transferred to a 1.5 ml tube. Then Buffer AE (100pi) was pipetted onto the DNeasy membrane. The mixture was incubated for5 min at room temperature (15-25° C.) and then centrifuged for 1 min(>8000 rpm) to elute.

The quality and quantity of the DNA was estimated by running samples onan agarose gel.

B. Cloning of 5′ Flanking Sequences of the Structural Gene

A modified inverse PCR method was used to clone 750 nucleotides of the5′ flanking sequences of the structural gene from SEQ ID NO:1. First,appropriate restriction enzymes were selected based on the restrictionsite in the known sequence fragment and the restriction sites distancedownstream of the 5′ flanking sequences. Two primers were designed basedon this known fragment. The forward primer was located downstream of thereverse primer. The reverse primer was located in the 3′ portion of theknown fragment.

The cloning procedure included the following steps:

The purified genomic DNA (5 μg) was digested with 20-40 units of theappropriate restriction enzyme (EcoRI and SpeI) in a 50 μl reactionmixture. An agarose gel electrophoresis with a 1/10 volume of thereaction mixture was performed to determine if the DNA was digested tocompletion. A direct ligation was performed after thorough digestion byligating overnight at 4° C. A reaction mixture of 200 μl containing 10μl of digested DNA and 0.2 μl of T4 DNA ligase (NEB) was ligatedovernight at 4° C. PCR on the ligation reaction was performed after anartificial small circular genome was obtained. PCR was performed with 10μl of ligation reaction and 2 primers from known fragments in twodifferent directions in 50 μl reaction mixture. A gradient PCR programwith annealing temperatures of 45-56° C. was applied.

Agarose gel electrophoresis was performed to check the PCR reaction. Thedesired band was cut from the gel and a QlAquick gel purification Kitfrom QIAGEN was used to purify the band. The purified PCR fragments wereligated into a pGEM-T Easy Vector (Promega, Madison, Wis.) followingmanufacturer's instructions. The transformed DNA plasmids were extractedby miniprep using SV Miniprep kit (Promega, Madison, Wis.) following themanufacturer's instructions. Plasmid DNA containing the insert wassequenced using a CEQ 2000 sequencer (Beckman, Fullerton, Calif.).Approximately 758 nt (nucleotides 1241-2009 of SEQ ID NO:1) of the 5′flanking sequence were cloned by the method described above.

C. Cloning of the Longer 5′ Flanking Sequences (SEQ ID NO:6; FIG. 5) ofthe Structural Gene

BD GenomeWalker Universal Kit (Clontech laboratories, Inc., PaloAlto,Calif.) was used for cloning additional 5′ flanking sequence of thestructural gene, D121-AA8 according to the manufacturer's user manual.The manufacturer's manual BD GenomeWalker August, 2004 is incorporatedhereby as reference. The size and purity of tobacco genomic DNA weretested by running samples on a 0.5% agarose gel. A total of 4 blunt-endreactions (DRA I, STU I, ECOR V, PVU II) were set up for tobacco 33library genome walking construction. After purification of the digestedDNAs, the digested genomic DNAs were ligated to the genome walkeradaptor. Primary PCR reactions were applied to the four digested DNAs byusing adaptor primer API and the gene specific primer from D121-AA8(CTCTATTGATACTAGCTGGTTTTGGAC; SEQ ID NO:19). The primary PCR productswere used directly as templates for the nested PCR. The adaptor nestedprimer provided by the kit and the nested primer from the known cloneD121-AA8 (SEQ ID NO:3) (GGAGGGAGAGTATAACTTACGGATTC; SEQ ID NO:20) wereused in the PCR reaction. PCR products were checked by running gelelectrophoreses. The desired bands were sliced out from the gel, and thePCR fragments were purified using QlAquick gel purification Kit fromQIAGEN. The purified PCR fragments were ligated into a pGEM-T EasyVector (Promega, Madison, Wis.) following manufacturer's instructions.The transformed DNA plasmids were extracted by miniprep using the SVMiniprep kit (Promega, Madison, Wis.) and following the manufacturer'sinstructions. Plasmid DNA containing the insert was sequenced using aCEQ 2000 sequencer (Beckman, Fullerton, Calif.). Another approximately853 nt of the 5′ flanking sequence, including nucleotides 399-1240 ofSEQ ID NO:1, were cloned by the method described above.

A second round of the genome walking was performed according to the samemethod with the difference that the following primers GWR1A(5′-AGTAACCGATTGCTCACGTTATCCTC-3′) (SEQ ID NO:21) and GWR2A(5′-CTCTATTCAACCCCACACGTAACTG-3′) (SEQ ID NO:22) were used. Anotherapproximately 398 nt of flanking sequence, including nucleotides 1-398of SEQ ID NO:1, were cloned by this method.

A search for regulatory elements revealed that, in addition to “TATA”box, “CAAT” boxes, and “GAGA” boxes, several MYB-like recognition sitesand organ specificity elements are present in the tobacco nicotinedemethylase promoter region. Putative elicitor responsive elements andnitrogen-regulated elements, identified using standard methods, are alsopresent in the promoter region.

D. Cloning of 3′ Flanking Sequences of the Structural Gene

BD GenomeWalker Universal Kit (Clontech laboratories, Inc., PaloAlto,Calif.) was used for cloning of 3′ flanking sequence of the structuralgene, D121-AA8 according to the manufacturer's user manual. The cloningprocedure is the same as describes in the preceding Section C of thisexample, except for the gene specific primers. The first primer wasdesigned from close to the end of D121-AA8 structural gene (5′-CTA AACTCT GGT CTG ATC CTG ATA CTT-3′) (SEQ ID NO:23). The nested primer wasdesigned further downstream of primer 1 of the D121-AA8 structural gene(CTA TAC GTA AGG TAA ATC CTG TGG AAC) (SEQ ID NO:24). The final PCRproducts were checked by gel electrophoreses. The desired bands wereexcised from the gel. The PCR fragments were purified using QlAquick gelpurification Kit from QIAGEN. The purified PCR fragments were ligatedinto a pGEM-T Easy Vector (Promega, Madison, Wis.) followingmanufacturer's instructions. The transformed DNA plasmids were extractedby miniprep using SV Miniprep kit (Promega, Madison, Wis.) followingmanufacturer's instructions. Plasmid DNA containing the insert wassequenced using a CEQ 2000 sequencer (Beckman, Fullerton, Calif.).Approximately 1617 nucleotides of additional 3′ flanking sequence(nucleotides 4731-6347 of SEQ ID NO:1) were cloned by the methoddescribed above. The nucleic acid sequence of the 3′UTR region is shownin FIG. 6.

WO 03/078577, WO 2004/035745, PCT/US/2004/034218, andPCT/US/2004/034065, and all other references, patents, patentapplication publications, and patent applications referred to herein areincorporated by reference herein to the same extent as if each of thesereferences, patents, patent application publications, and patentapplications were separately incorporated by reference herein.

Numerous modifications and variations in practice of the invention areexpected to occur to those skilled in the art upon consideration of theforegoing detailed description of the invention. Consequently, suchmodifications and variations are intended to be included within thescope of the following claims.

What is claimed is:
 1. A tobacco leaf from a transgenic tobacco plantcomprising a vector, said vector comprising a nucleotide sequence having100% sequence identity to 25 or more contiguous nucleotides of SEQ IDNO:1, wherein the transgenic tobacco plant exhibits reduced expressionof the nicotine demethylase polypeptide encoded by SEQ ID NO:1 relativeto a non-transgenic tobacco plant.
 2. The tobacco leaf of claim 1, saidnucleotide sequence having at least 91% sequence identity to 100 or morecontiguous nucleotides of SEQ ID NO:1.
 3. The tobacco leaf of claim 2,said nucleotide sequence having at least 91% sequence identity to 250 ormore contiguous nucleotides of SEQ ID NO:1.
 4. The tobacco leaf of claim3, said nucleotide sequence having at least 91% sequence identity to 500or more contiguous nucleotides of SEQ ID NO:1.
 5. The tobacco leaf ofclaim 1, said nucleotide sequence having at least 91% sequence identityto 50 or more contiguous nucleotides of nucleotides 3947-4562 of SEQ IDNO:1.
 6. The tobacco leaf of claim 5, said nucleotide sequence having100% sequence identity to 50 or more contiguous nucleotides ofnucleotides 3947-4562 of SEQ ID NO:1.
 7. The tobacco leaf of claim 5,said nucleotide sequence having 91% sequence identity to 100 or morecontiguous nucleotides of nucleotides 3947-4562 of SEQ ID NO:1.
 8. Thetobacco leaf of claim 7, said nucleotide sequence having 100% sequenceidentity to 100 or more contiguous nucleotides of nucleotides 3947-4562of SEQ ID NO:1.
 9. The tobacco leaf of claim 8, said nucleotide sequencehaving 100% sequence identity to 250 or more contiguous nucleotides ofnucleotides 3947-4562 of SEQ ID NO:1.
 10. The tobacco leaf of claim 1,said nucleotide sequence having 91% sequence identity to 50 or morecontiguous nucleotides of nucleotides 2010-2949 of SEQ ID NO:1.
 11. Thetobacco leaf of claim 10, said nucleotide sequence having 100% sequenceidentity to 50 or more contiguous nucleotides of nucleotides 2010-2949of SEQ ID NO:1.
 12. The tobacco leaf of claim 10, said nucleotidesequence having 91% sequence identity to 100 or more contiguousnucleotides of nucleotides 2010-2949 of SEQ ID NO:1.
 13. The tobaccoleaf of claim 12, said nucleotide sequence having 100% sequence identityto 100 or more contiguous nucleotides of nucleotides 2010-2949 of SEQ IDNO:1.
 14. The tobacco leaf of claim 13, said nucleotide sequence having100% sequence identity to 250 or more contiguous nucleotides ofnucleotides 2010-2949 of SEQ ID NO:1.
 15. The tobacco leaf of claim 1,wherein said leaf is a cured leaf.
 16. A cured tobacco plant componentfrom a transgenic tobacco plant comprising a vector, said vectorcomprising a nucleotide sequence having 100% sequence identity to 25 ormore contiguous nucleotides of SEQ ID NO:1, said cured tobacco plantcomponent exhibiting a reduced amount of the nicotine demethylasepolypeptide encoded by SEQ ID NO:1 relative to the amount in acorresponding cured tobacco plant component obtained from a wild-typenon-converter tobacco plant.
 17. The cured tobacco plant component ofclaim 16, wherein said cured tobacco plant component is a tobacco leaf.18. The cured tobacco plant component of claim 17, wherein said curedtobacco plant component has a reduced level of nornicotine relative tothe level in a cured tobacco plant component obtained from a wild-typenon-converter tobacco plant.
 19. The cured tobacco plant component ofclaim 17, wherein said cured tobacco plant component is from a darktobacco, Burley tobacco, flue-cured tobacco, air-cured tobacco, ororiental tobacco plant.
 20. A tobacco product comprising the curedtobacco plant component of claim
 17. 21. The tobacco product of claim20, wherein said tobacco product is smokeless tobacco.
 22. The tobaccoproduct of claim 21, wherein said tobacco product is a moist or drysnuff.
 23. The tobacco product of claim 21, wherein said tobacco productis a chewing tobacco.
 24. The tobacco product of claim 20, wherein saidtobacco product is a cigarette.
 25. The tobacco product of claim 20,wherein said tobacco product is a cigar or cigarillo.